Sphingosine-1-phosphate lyase polypeptides, polynucleotides and modulating agents and methods of use therefor

ABSTRACT

Compositions, methods and kits for diagnosing and treating cancer are provided. Therapeutic compositions may comprise agents that modulate the expression or activity of a sphingosine-1-phosphate lyase (SPL). Such compositions may be administered to a mammal afflicted with cancer. Diagnostic methods and kits may employ an agent suitable for detecting alterations in endogenous SPL. Such methods and kits may be used to detect the presence of a cancer or to evaluate the prognosis of a known disease. SPL polypeptides, polynucleotides and antibodies are also provided.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to cancer detection andtherapy. The invention is more particularly related tosphingosine-1-phosphate lyase polynucleotides and polypeptides, and toagents that modulate the expression and/or activity of suchpolypeptides. Such agents may be used, for example, to diagnose and/ortreat cancers such as breast and colon cancer.

[0003] 2. Description of the Related Art

[0004] Breast cancer is a significant health problem for women in theUnited States and throughout the world. Although advances have been madein detection and treatment of the disease, breast cancer remains themost common form of cancer, and the second leading cause of cancerdeath, in American women. Among African-American women and women between15 and 54 years of age, breast cancer is the leading cause of cancerdeath. One out of every eight women in the United States will developbreast cancer, a risk which has increased 52% during 1950-1990. In 1994,it is estimated that 182,000 new cases of female breast cancer werediagnosed, and 46,000 women died from the disease.

[0005] No vaccine or other universally successful method for theprevention or treatment of breast cancer is currently available.Management of the disease currently relies on a combination of earlydiagnosis (through routine breast screening procedures) and aggressivetreatment, which may include one or more of a variety of treatments suchas surgery, radiotherapy, chemotherapy and hormone therapy. The courseof treatment for a particular breast cancer is often selected based on avariety of prognostic parameters, including an analysis of specifictumor markers. However, the use of established markers often leads to aresult that is difficult to interpret.

[0006] With current therapies, tumor invasiveness and metastasis is acritical determinant in the outcome for breast cancer patients. Althoughthe five year survival for women diagnosed with localized breast canceris about 90%, the five year survival drops to 18% for women whosedisease has metastasized. Present therapies are inadequate forinhibiting tumor invasiveness for the large population of women withthis severe disease.

[0007] Colon cancer is the second most frequently diagnosed malignancyin the United States as well as the second most common cause of cancerdeath. The five-year survival rate for patients with colorectal cancerdetected in an early localized stage is 92%; unfortunately, only 37% ofcolorectal cancer is diagnosed at this stage. The survival rate drops to64% if the cancer is allowed to spread to adjacent organs or lymphnodes, and to 7% in patients with distant metastases.

[0008] The prognosis of colon cancer is directly related to the degreeof penetration of the tumor through the bowel wall and the presence orabsence of nodal involvement, consequently, early detection andtreatment are especially important. Currently, diagnosis is aided by theuse of screening assays for fecal occult blood, sigmoidoscopy,colonoscopy and double contrast barium enemas. Treatment regimens aredetermined by the type and stage of the cancer, and include surgery,radiation therapy and/or chemotherapy. Recurrence following surgery (themost common form of therapy) is a major problem and is often theultimate cause of death. In spite of considerable research intotherapies for the disease, colon cancer remains difficult to diagnoseand treat. In spite of considerable research into therapies for theseand other cancers, colon cancer remains difficult to diagnose and treateffectively. Accordingly, improvements are needed in the treatment,diagnosis and prevention of breast and colon cancer. The presentinvention fulfills this need and further provides other relatedadvantages.

BRIEF SUMMARY OF THE INVENTION

[0009] Briefly stated, the present invention provides compositions andmethods for the diagnosis and therapy of cancer. Within one aspect, thepresent invention provides isolated polynucleotides comprising asequence selected from the group consisting of: (a) a sequence shown inSEQ ID NO:15; (b) nucleotide sequences that hybridize to apolynucleotide complementary to a sequence shown in SEQ ID NO:15 undermoderately stringent conditions, wherein the nucleotide sequences encodepolypeptides having sphingosine-1-phosphate lyase activity; and (c)nucleotide sequences that encode a polypeptide encoded by a sequenceshown in SEQ ID NO:15.

[0010] Within a related aspect, an isolated polynucleotide is providedthat encodes a polypeptide shown in SEQ ID NO:16, or a variant of such apolypeptide that has sphingosine-1-phosphate lyase activity. Recombinantexpression vectors comprising any of the foregoing polynucleotides, andhost cells transformed or transfected with such expression vectors, arealso provided.

[0011] Within further aspects, SPL polypeptides are provided. Suchpolypeptides may be encoded by any of the foregoing polynucleotides.Alternatively, a polypeptide may comprise an amino acid sequence shownin SEQ ID NO:16, or a variant thereof, wherein the polypeptide hassphingosine-1-phosphate lyase activity.

[0012] Within a further aspect, the present invention provides isolatedpolynucleotides comprising at least 100 nucleotides complementary to asequence shown in SEQ ID NO:15.

[0013] Within other aspects, methods are provided for preparing asphingosine-1-phosphate lyase, comprising culturing a host celltransformed or transfected with a polynucleotide as described aboveunder conditions promoting expression of the polynucleotide andrecovering a sphingosine-1-phosphate lyase.

[0014] In further aspects, the present invention provides methods foridentifying an agent that modulates sphingosine-1-phosphate lyaseactivity. In one such aspect, the method comprises: (a) contacting acandidate agent with a polypeptide comprising a sequence shown in SEQ IDNO:16, or a variant of such a sequence having sphingosine-1-phosphatelyase activity, wherein the step of contacting is carried out underconditions and for a time sufficient to allow the candidate agent tointeract with the polypeptide; and (b) subsequently measuring theability of the polypeptide to degrade sphingosine-1-phosphate or aderivative thereof, relative to an ability in the absence of candidateagent. The step of contacting may be performed by incubating a cellexpressing the polypeptide with the candidate modulator, and the step ofmeasuring the ability to degrade sphingosine-1-phosphate may beperformed using an in vitro assay and a cellular extract.

[0015] The present invention further provides pharmaceuticalcompositions comprising an agent that modulates sphingosine-1-phosphatelyase activity in combination with a pharmaceutically acceptablecarrier. Such agents preferably increase sphingosine-1-phosphate lyaseactivity. Such inhibition may be achieved by increasing expression of anendogenous SPL gene, or by increasing the ability of an endogenous SPLto degrade sphingosine-1-phosphate. Within certain preferredembodiments, a modulating agent comprises a polynucleotide or anantibody or an antigen-binding fragment thereof.

[0016] Within still further aspects, the present invention providesmethods for modulating sphingosine-1-phosphate activity, comprisingcontacting a sphingosine-1-phosphate lyase with an effective amount ofan agent that modulates sphingosine-1-phosphate lyase activity, whereinthe step of contacting is performed under conditions and for a timesufficient to allow the agent and the sphingosine-1-phosphate lyase tointeract. To modulate sphingosine-1-phosphate lyase activity in a cell,a cell expressing sphingosine-1-phosphate may be contacted with such anagent.

[0017] Within related aspects, the present invention provides methodsfor inhibiting the growth of a cancer cell, comprising contacting acancer cell with an agent that increases sphingosine-1-phosphate lyaseactivity. In a preferred embodiment, the cancer cell is a breast cancercell.

[0018] The present invention also provides methods for inhibiting thedevelopment and/or metastasis of a cancer in a mammal, comprisingadministering to a mammal an agent that increasessphingosine-1-phosphate lyase activity. Within certain embodiments, anagent may comprise, or be linked to, a targeting component, such as ananti-tumor antibody or a component that binds to an estrogen receptor.

[0019] Within other aspects, methods for diagnosing cancer in a mammalare provided, comprising detecting an alteration in an endogenoussphingosine-1-phosphate lyase gene in a sample obtained from a mammal,and therefrom diagnosing a cancer in the mammal. In certain embodimentsthe cancer is breast or colon cancer and the sample is a breast tumorbiopsy.

[0020] In related aspects, the present invention provides methods forevaluating a cancer prognosis, comprising determining the presence orabsence of an alteration in an endogenous sphingosine-1-phosphate lyasegene in a sample obtained from a mammal afflicted with cancer, andtherefrom determining a prognosis.

[0021] The present invention further provides isolated antibodies thatbind to a polypeptide having a sequence shown in SEQ ID NO:16. Suchantibodies may be polyclonal or monoclonal, and may increase the abilityof a polypeptide having a sequence shown in SEQ ID NO:16 degradesphingosine-1-phosphate.

[0022] In still further aspects, the present invention provides methodsfor detecting sphingosine-1-phosphate lyase in a sample, comprising: (a)contacting a sample with an antibody as described above under conditionsand for a time sufficient to allow the antibody to bind tosphingosine-1-phosphate lyase; and (b) detecting in the sample thepresence of sphingosine-1-phosphate lyase bound to the antibody.

[0023] Kits for use in the above methods are also provided. A kit fordetecting sphingosine-1-phosphate lyase in a sample comprises anantibody as described above and a buffer or detection reagent. A kit fordetecting an alteration in a sphingosine-1-phosphate gene in a samplecomprises a polynucleotide and a detection reagent.

[0024] The present invention further provides for a homozygous nullmutant Drosophila melanogaster fly line the genome of which comprises aP-element transposon insertion in the coding region of the sphingosinephosphate lyase (SPL) gene wherein said gene encodes the sequence setforth in SEQ ID NO:16, and wherein said fly line has a flightlessphenotype. In a related embodiment, the homozygous mutant fliesdemonstrate abnormal developmental patterning of thoracic muscles of theT2 segment.

[0025] The present invention also provides methods for testing an agentcapable of inhibiting the development and/or metastasis of a cancer in amammal, comprising contacting SPL mutant Drosophila progeny with growthmedium comprising a test agent suspected of inhibiting mammaliansphingosine kinase, and detecting the restoration of flight ability inthe progeny. In a related embodiment, the homozygous mutant flies usedin this method demonstrate abnormal developmental patterning of thoracicmuscles of the T2 segment.

[0026] The present invention further provides for methods fordetermining the presence of a cancer in a patient, comprising the stepsof: (a) obtaining a biological sample from the patient; (b) contactingthe biological sample with at least one oligonucleotide that is at leastpartially complementary to the sequence set forth in SEQ ID NO:7; (c)detecting in the sample an amount of said oligonucleotide thathybridizes to the polynucleotide; and comparing the amount ofoligonucleotide that hybridizes to the polynucleotide to a predeterminedcut-off value, and therefrom determining the presence of the cancer inthe patient.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

[0027] SEQ ID NO:1 is the determined cDNA sequence of S. cerevisiae SPL

[0028] SEQ ID NO:2 is the amino acid sequence of S. cerevisiae SPLencoded by the polynucleotide sequence set forth in SEQ ID NO:1

[0029] SEQ ID NO:3 is the determined cDNA sequence of C. elegans SPL

[0030] SEQ ID NO:4 is the amino acid sequence of C. elegans SPL encodedby the polynucleotide sequence set forth in SEQ ID NO:3

[0031] SEQ ID NO:5 is the determined cDNA sequence of the mouse SPL

[0032] SEQ ID NO:6 is the amino acid sequence of mouse SPL encoded bythe polynucleotide sequence set forth in SEQ ID NO:5

[0033] SEQ ID NO:7 is the determined cDNA sequence of the full-lengthhuman SPL

[0034] SEQ ID NO:8 is the amino acid sequence of human SPL encoded bythe polynucleotide sequence set forth in SEQ ID NO:7

[0035] SEQ ID NO:9 is the determined cDNA sequence of a human SPL with adeletion

[0036] SEQ ID NO:10 is the amino acid sequence of a human SPL with adeletion, encoded by the polynucleotide sequence set forth in SEQ IDNO:9

[0037] SEQ ID NO:11 is the amino acid sequence of C. elegans SPL encodedby the polynucleotide sequence set forth in SEQ ID NO: 12

[0038] SEQ ID NO:12 is the determined cDNA sequence of a C. elegans SPL

[0039] SEQ ID NO:13 is a PCR primer

[0040] SEQ ID NO:14 is a PCR primer

[0041] SEQ ID NO:15 is the determined cDNA sequence encoding theDrosophila melanogaster SPL

[0042] SEQ ID NO:16 is the amino acid sequence of the Drosophilamelanogaster SPL, encoded by the cDNA sequence set forth in SEQ ID NO:15

[0043] SEQ ID NO:17 is the determined cDNA sequence of a human SPL asset forth in Genbank Accession No: AF144638.

[0044] SEQ ID NO:18 is the amino acid sequence of a human SPL encoded bythe polynucleotide sequence provided in SEQ ID NO:17.

[0045] SEQ ID NO:19 is the amino acid sequence of a first Drosophilamelanogaster SK protein.

[0046] SEQ ID NO:20 is the amino acid sequence of a second Drosophilamelanogaster SK protein.

[0047] SEQ ID NO:21 is the amino acid sequence of a human SK protein.

DETAILED DESCRIPTION OF THE INVENTION

[0048] As noted above, the present invention is generally directed tocompositions and methods for the diagnosis and therapy of cancers suchas breast cancer. The invention is more particularly related tosphingosine-1-phosphate lyase (SPL) polypeptides, which have the abilityto cleave sphingosine-1-phosphate into inactive metabolites, and topolynucleotides encoding such polypeptides. Sphingosine-1-phosphate(S-1-P) is an endogenous sphingolipid metabolite present in mostmammalian cells and in serum. Like other sphingolipid metabolites suchas ceramide and sphingosine, S-1-P participates in specific signaltransduction pathways. The results of S-1-P signaling are diverse anddependent upon the cell type being examined. However, many of theeffects of S-1-P signaling, which include promotion of cellularproliferation, enhancement of migration, inhibition of apoptosis andstimulation of angiogenesis, influence the transformation, growth, drugresistance, vascularity and metastatic capacity of cancer cells. Thegene encoding the enzyme responsible for S-1-P synthesis is sphingosinekinase, SK, and S-1-P degradation is sphingosine phosphate lyase, SPLand S-1-P phosphatase, S-1-PP. Several observations support the notionthat SPL may be a cancer related gene. First, altered expression of SPLin human tumors compared to corresponding normal tissue from the samepatient has been shown. Second, human SPL maps to 10q21, a chromosomalregion frequently deleted in a variety of human cancers. Taken together,these observations raise the possibility that SPL may be potentiallyeffective targets for pharmacological intervention in the treatment ofcancer.

[0049] Agents that decrease the expression or activity of endogenous SPLpolypeptides are encompassed by the present invention. Such modulatingagents may be identified using methods described herein and used, forexample, in cancer therapy. It has also been found, within the contextof the present invention, that the detection of alterations in anendogenous SPL sequence can be used to diagnose cancer, and to assessthe prognosis for recovery. The present invention further provides suchdiagnostic methods and kits.

[0050] As used herein, the term “polypeptide” encompasses amino acidchains of any length, including full length endogenous (i.e., native)SPL proteins and variants of endogenous sequences. “Variants” arepolypeptides that differ in sequence from a native SPL only insubstitutions, deletions and/or other modifications, such that thevariant retains SPL activity, which may be determined using arepresentative method described herein SPL polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity along its length, to an SPL polypeptidesequence set forth herein. Within an SPL polypeptide variant, amino acidsubstitutions are preferably made at no more than 50% of the amino acidresidues in the native polypeptide, and more preferably at no more than25% of the amino acid residues. Such substitutions are preferablyconservative. A conservative substitution is one in which an amino acidis substituted for another amino acid that has similar properties, suchthat one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. In general, the following amino acids representconservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr;(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg,his; and (5) phe, tyr, trp, his. Substitutions, deletions and/or aminoacid additions may be made at any location(s) in the polypeptide,provided that the modification does not diminish the SPL activity of thevariant. Thus, a variant may comprise only a portion of a native SPLsequence. In addition, or alternatively, variants may contain additionalamino acid sequences (such as, for example, linkers, tags and/orligands), preferably at the amino and/or carboxy termini. Such sequencesmay be used, for example, to facilitate purification, detection orcellular uptake of the polypeptide.

[0051] When comparing polypeptide sequences, two sequences are said tobe “identical” if the sequence of amino acids in the two sequences isthe same when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

[0052] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Saitou, N. Nei, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0053] Alternatively, optimal alignment of sequences for comparison maybe conducted by the local identity algorithm of Smith and Waterman(1981) Add. APL. Math 2:482, by the identity alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.USA 85: 2444, by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

[0054] One preferred example of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides and polypeptides of theinvention. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. For aminoacid sequences, a scoring matrix can be used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

[0055] The SPL activity of an SPL polypeptide or variant thereof maygenerally be assessed using an in vitro assay that detects thedegradation of labeled substrate (i.e., sphingosine-1-phosphate, or aderivative thereof). Within such assays, pyridoxal 5′-phosphate is arequirement for SPL activity. In addition, the reaction generallyproceeds optimally at pH 7.4-7.6 and requires chelators due tosensitivity toward heavy metal ions. The substrate should be a D-erythroisomer, but in derivatives of sphingosine-1-phosphate the type and chainlength of sphingoid base may vary. In general, an assay as described byVan Veldhoven and Mannaerts, J. Biol. Chem. 266:12502-07, 1991 may beemployed. Briefly, a solution (e.g., a cellular extract) containing thepolypeptide may be incubated with 40 μM substrate at 37° C. for 1 hourin the presence of, for example, 50 mM sucrose, 100 mM K-phosphatebuffer pH 7.4, 25 mM NaF, 0.1% (w/v) Triton X-100, 0.5 mM EDTA, 2 mMDTT, 0.25 mM pyridoxal phosphate. Reactions may then be terminated andanalyzed by thin-layer chromatography to detect the formation of labeledfatty aldehydes and further metabolites. In general, a polypeptide hasSPL activity if, within such an assay: (1) the presence of 2-50 μgpolypeptide (or 0.1-10 mg/mL) results in a statistically significantincrease in the level of substrate degradation, preferably a two-foldincrease, relative to the level observed in the absence of polypeptide;and (2) the increase in the level of substrate degradation is pyridoxal5′-phosphate dependent.

[0056] Within certain embodiments, an in vitro assay for SPL activitymay be performed using cellular extracts prepared from cells thatexpress the polypeptide of interest. Preferably, in the absence of agene encoding an SPL polypeptide, such cells do not produce asignificant amount of endogenous SPL (i.e., a cellular extract shouldnot contain a detectable increase in the level of SPL, as compared tobuffer alone without extract). It has been found, within the context ofthe present invention, that yeast cells containing deletion of the SPLgene (BST1) are suitable for use in evaluating the SPL activity of apolypeptide. bst1Δ cells can be generated from S. cerevisiae usingstandard techniques, such as PCR, as described herein. A polypeptide tobe tested for SPL activity may then be expressed in bst1Δ cells, and thelevel of SPL activity in an extract containing the polypeptide may becompared to that of an extract prepared from cells that do not expressthe polypeptide. For such a test, a polypeptide is preferably expressedon a high-copy yeast vector (such as pYES2, which is available fromInvitrogen) yielding more than 20 copies of the gene per cell. Ingeneral, a polypeptide has SPL activity if, when expressed using such avector in a bst1Δ cell, a cellular extract results in a two-foldincrease in substrate degradation over the level observed for an extractprepared from cells not expressing the polypeptide.

[0057] A further test for SPL activity may be based upon functionalcomplementation in the bst1Δ strain. It has been found, within thecontext of the present invention, that bst1Δ cells are highly sensitiveto D-erythro-sphingosine. In particular, concentrations as low as 10 μMsphingosine completely inhibit the growth of bst1Δ cells. Such a levelof sphingosine has no effect on the growth of wildtype cells. Apolypeptide having SPL activity as provided above significantlydiminishes (i.e., by at least two fold) the sphingosine sensitivity whenexpressed on a high-copy yeast vector yielding more than 20 copies ofthe gene per cell.

[0058] In general, SPL polypeptides, and polynucleotides encoding suchpolypeptides, may be prepared using any of a variety of techniques thatare well known in the art. For example, a DNA sequence encoding nativeSPL may be prepared by amplification from a suitable cDNA or genomiclibrary using, for example, polymerase chain reaction (PCR) orhybridization techniques. Libraries may generally be prepared andscreened using methods well known to those of ordinary skill in the art,such as those described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989. cDNA libraries may be prepared from any of a variety ofsources known to contain enzymes having SPL activity. SPL activity isubiquitous with regard to species and mammalian tissues, with theexception of platelets, in which SPL activity is notably absent. In rattissues, the highest levels of activity have been demonstrated inintestinal mucosa, liver and Harderian gland, with low activity inskeletal muscle and heart. Activity has also been demonstrated in anumber of human (hepatoma cell line HB 8065, cervical carcinoma HeLa),mouse (hepatoma line BW1, mouse embryo 3T3-L1, Swiss 3T3 cells) andother cell lines, as well as in human cultured fibroblasts. PreferredcDNA libraries may prepared from human liver, intestine or brain tissuesor cells. Other libraries that may be employed will be apparent to thoseof ordinary skill in the art. Primers for use in amplification may bereadily designed based on the sequence of a native SPL polypeptide orpolynucleotide, as provided herein.

[0059] Alternatively, an endogenous SPL gene may be identified using ascreen for cDNAs that complement the BST1 deletion in yeast. A cDNAexpression library may be generated using a regulatable yeast expressionvector (e.g., pYES, which is availablve from Invitrogen, Inc.) andstandard techniques. A yeast bst1Δ strain may then be transformed withthe cDNA library, and endogenous cDNAs having the ability tofunctionally complement the yeast lyase defect (ie., restore the abilityto grow in the presence of D-erythro-sphingosine) may be isolated.

[0060] An endogenous SPL gene may also be identified based oncross-reactivity of the protein product with anti-SPL antibodies, whichmay be prepared as described herein. Such screens may generally beperformed using standard techniques (see Huynh et al., “Construction andScreening cDNA Libraries in λgt11,” in D. M. Glover, ed., DNA Cloning: APractical Approach, 1:49-78, 1984 (IRL Press, Oxford)).

[0061] Polynucleotides encompassed by the present invention include DNAand RNA molecules that comprise an endogenous SPL gene sequence. Suchpolynucleotides include those that comprise a sequence recited in anyone of SEQ ID NOs:1-16. Also encompassed are other polynucleotides thatencode an SPL amino acid sequence encoded by such polynucleotides, aswell as polynucleotides that encode variants of a native SPL sequencethat retain SPL activity. Polynucleotides that are substantiallyhomologous to a sequence complementary to an endogenous SPL gene arealso within the scope of the present invention. “Substantial homology,”as used herein refers to polynucleotides that are capable of hybridizingunder moderately stringent conditions to a polynucleotide complementaryto an SPL polynucleotide sequence provided herein, provided that theencoded SPL polypeptide variant retains SPL activity. Suitablemoderately stringent conditions include prewashing in a solution of5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50-65° C., 5×SSC,overnight; followed by washing twice at 65° C. for 20 minutes with eachof 2×, 0.5× and 0.2×SSC containing 0.1% SDS. Nucleotide sequences that,because of code degeneracy, encode a polypeptide encoded by any of theabove sequences are also encompassed by the present invention.

[0062] Polypeptides of the present invention may be prepared byexpression of recombinant DNA encoding the polypeptide in cultured hostcells. Preferably, the host cells are bacteria, yeast, insect ormammalian cells, and more preferably the host cells are S. cerevisiaebst1Δ cells. The recombinant DNA may be cloned into any expressionvector suitable for use within the host cell and transfected into thehost cell using techniques well known to those of ordinary skill in theart. A suitable expression vector contains a promoter sequence that isactive in the host cell. A tissue-specific or conditionally activepromoter may also be used. Preferred promoters express the polypeptideat high levels.

[0063] Optionally, the construct may contain an enhancer, atranscription terminator, a poly(A) signal sequence, a bacterial ormammalian origin of replication and/or a selectable marker, all of whichare well known in the art. Enhancer sequences may be included as part ofthe promoter region or separately. Transcription terminators aresequences that stop RNA polymerase-mediated transcription. The poly(A)signal may be contained within the termination sequence or incorporatedseparately. A selectable marker includes any gene that confers aphenotype on the host cell that allows transformed cells to beidentified. Such markers may confer a growth advantage under specifiedconditions. Suitable selectable markers for bacteria are well known andinclude resistance genes for ampicillin, kanamycin and tetracycline.Suitable selectable markers for mammalian cells include hygromycin,neomycin, genes that complement a deficiency in the host (e.g.,thymidine kinase and TK⁻cells) and others well known in the art. Foryeast cells, one suitable selectable marker is URA3, which confers theability to grow on medium without uracil.

[0064] DNA sequences expressed in this manner may encode a native SPLpolypeptide (e.g., human), or may encode portions or other variants ofnative SPL polypeptide. DNA molecules encoding variants of a native SPLmay generally be prepared using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis, and sections of theDNA sequence may be removed to permit preparation of truncatedpolypeptides.

[0065] To generate cells that express a polynucleotide encoding an SPLpolypeptide, cells may be transfected, transformed or transduced usingany of a variety of techniques known in the art. Any number oftransfection, transformation, and transduction protocols known to thosein the art may be used, for example those outlined in Current Protocolsin Molecular Biology, John Wiley & Sons, New York. N.Y., or in numerouskits available commercially (e.g., Invitrogen Life Technologies,Carlsbad, Calif.). Such techniques may result in stable transformants ormay be transient. One suitable transfection technique iselectroporation, which may be performed on a variety of cell types,including mammalian cells, yeast cells and bacteria, using commerciallyavailable equipment. Optimal conditions for electroporation (includingvoltage, resistance and pulse length) are experimentally determined forthe particular host cell type, and general guidelines for optimizingelectroporation may be obtained from manufacturers. Other suitablemethods for transfection will depend upon the type of cell used (e.g.,the lithium acetate method for yeast), and will be apparent to those ofordinary skill in the art. Following transfection, cells may bemaintained in conditions that promote expression of the polynucleotidewithin the cell. Appropriate conditions depend upon the expressionsystem and cell type, and will be apparent to those skilled in the art.

[0066] SPL polypeptides may be expressed in transfected cells byculturing the cell under conditions promoting expression of thetransfected polynucleotide. Appropriate conditions will depend on thespecific host cell and expression vector employed, and will be readilyapparent to those of ordinary skill in the art. For commerciallyavailable expression vectors, the polypeptide may generally be expressedaccording to the manufacturer's instructions. For certain purposes,expressed polypeptides of this invention may be isolated insubstantially pure form. Preferably, the polypeptides are isolated to apurity of at least 80% by weight, more preferably to a purity of atleast 95% by weight, and most preferably to a purity of at least 99% byweight. In general, such purification may be achieved using, forexample, the standard techniques of ammonium sulfate fractionation,SDS-PAGE electrophoresis, and/or affinity chromatography.

[0067] The present invention further provides antibodies that bind to anSPL polypeptide. Antibodies may function as modulating agents (asdiscussed further below) to inhibit or block SPL activity in vivo.Alternatively, or in addition, antibodies may be used within screens forendogenous SPL polypeptides or modulating agents, for purification ofSPL polypeptides, for assaying the level of SPL within a sample and/orfor studies of SPL expression. Such antibodies may be polyclonal ormonoclonal, and are generally specific for one or more SPL polypeptidesand/or one or more variants thereof. Within certain preferredembodiments, antibodies are polyclonal.

[0068] Antibodies may be prepared by any of a variety of techniquesknown to those of ordinary skill in the art (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In one such technique, an immunogen comprising an SPL polypeptide orantigenic portion thereof is initially injected into a suitable animal(e.g., mice, rats, rabbits, sheep and goats), preferably according to apredetermined schedule incorporating one or more booster immunizations.The use of rabbits is preferred. To increase immunogenicity, animmunogen may be linked to, for example, glutaraldehyde or keyholelimpet hemocyanin (KLH). Following injection, the animals are bledperiodically to obtain post-immune serum containing polyclonal anti-SPLantibodies. Polyclonal antibodies may then be purified from suchantisera by, for example, affinity chromatography using an SPLpolypeptide or antigenic portion thereof coupled to a suitable solidsupport. Such polyclonal antibodies may be used directly for screeningpurposes and for Western blots.

[0069] More specifically, an adult rabbit (e.g., NZW) may be immunizedwith 10 μg purified (e.g., using a nickel-column) SPL polypeptideemulsified in complete Freund's adjuvant (1:1 v/v) in a volume of 1 mL.Immunization may be achieved via injection in at least six differentsubcutaneous sites. For subsequent immunizations, 5 μg of an SPLpolypeptide may be emulsified in in complete Freund's adjuvant andinjected in the same manner. Immunizations may continue until a suitableserum antibody titer is achieved (typically a total of about threeimmunizations). The rabbit may be bled immediately before immunizationto obtain pre-immune serum, and then 7-10 days following eachimmunization.

[0070] For certain embodiments, monoclonal antibodies may be desired.Monoclonal antibodies may be prepared, for example, using the techniqueof Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, andimprovements thereto. Briefly, these methods involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity (ie., reactivity with the polypeptide of interest). Suchcell lines may be produced, for example, from spleen cells obtained froman animal immunized as described above. The spleen cells are thenimmortalized by, for example, fusion with a myeloma cell fusion partner,preferably one that is syngeneic with the immunized animal. For example,the spleen cells and myeloma cells may be combined with a nonionicdetergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

[0071] Monoclonal antibodies may be isolated from the supernatants ofgrowing hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host, such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction.

[0072] As noted above, the present invention provides agents thatmodulate, preferably inhibit, the expression (transcription ortranslation), stability and/or activity of an SPL polypeptide. Toidentify such a modulating agent, any of a variety of screens may beperformed. Candidate modulating agents may be obtained using well knowntechniques from a variety of sources, such as plants, fungi or librariesof chemicals, small molecules or random peptides. Antibodies that bindto an SPL polypeptide, and anti-sense polynucleotides that hybridize toa polynucleotides that encodes an SPL, may be candidate modulatingagents. Preferably, a modulating agent has a minimum of side effects andis non-toxic. For some applications, agents that can penetrate cells arepreferred.

[0073] Screens for modulating agents that decrease SPL expression orstability may be readily performed using well known techniques thatdetect the level of SPL protein or mRNA. Suitable assays include RNAseprotection assays, in situ hybridization, ELISAs, Northern blots andWestern blots. Such assays may generally be performed using standardmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). Forexample, to detect mRNA encoding SPL, a nucleic acid probe complementaryto all or a portion of the SPL gene sequence may be employed in aNorthern blot analysis of mRNA prepared from suitable cells.Alternatively, real-time PCR can also be used to detect levels of mRNAencoding SPL (see Gibson et al., Genome Research 6:995-1001, 1996; Heidet al., Genome Research 6:986-994, 1996). The first-strand cDNA to beused in the quantitative real-time PCR is synthesized from 20 μg oftotal RNA that is first treated with DNase I (e.g., Amplification Grade,Gibco BRL Life Technology, Gaitherburg, Md.), using Superscript ReverseTranscriptase (RT) (e.g., Gibco BRL Life Technology, Gaitherburg, Md.).Real-time PCR is performed, for example, with a GeneAmp™ 5700 sequencedetection system (PE Biosystems, Foster City, Calif.). The 5700 systemuses SYBR™ green, a fluorescent dye that only intercalates into doublestranded DNA, and a set of gene-specific forward and reverse primers.The increase in fluorescence is monitored during the whole amplificationprocess. The optimal concentration of primers is determined using acheckerboard. The PCR reaction is performed in 25 μl volumes thatinclude 2.5 μl of SYBR green buffer, 2 μl of cDNA template and 2.5 μleach of the forward and reverse primers for the SPL gene, or other geneof interest. The cDNAs used for RT reactions are diluted approximately1:10 for each gene of interest and 1:100 for the β-actin control. Inorder to quantitate the amount of specific cDNA (and hence initial mRNA)in the sample, a standard curve is generated for each run using theplasmid DNA containing the gene of interest. Standard curves aregenerated using the Ct values determined in the real-time PCR which arerelated to the initial cDNA concentration used in the assay. Standarddilution ranging from 20-2×10⁶ copies of the SPL gene or other gene ofinterest are used for this purpose. In addition, a standard curve isgenerated for β-actin ranging from 200 fg-2000 fg. This enablesstandardization of the initial RNA content of a sample to the amount ofβ-actin for comparison purposes. The mean copy number for each sampletested is normalized to a constant amount of β-actin, allowing theevaluation of the observed expression levels of SPL or other gene ofinterest.

[0074] To detect SPL protein, a reagent that binds to the protein(typically an antibody, as described herein) may be employed within anELISA or Western assay. Following binding, a reporter group suitable fordirect or indirect detection of the reagent is employed (i.e., thereporter group may be covalently bound to the reagent or may be bound toa second molecule, such as Protein A, Protein G, immunoglobulin orlectin, which is itself capable of binding to the reagent). Suitablereporter groups include, but are not limited to, enzymes (e.g.,horseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Suchreporter groups may be used to directly or indirectly detect binding ofthe reagent to a sample component using standard methods known to thoseof ordinary skill in the art.

[0075] To use such assays for identifying a modulating agent, the levelof SPL protein or mRNA may be evaluated in cells treated with one ormore candidate modulating agents. An increase or decrease in SPL levelsmay be measured by evaluating the level of SPL mRNA and/or protein inthe presence and absence of candidate modulating agent. For example, anantisense modulating agent may be evaluated by assaying the effect onSPL levels. Suitable cells for use in such assays include the breastcancer cell lines MCF-7 (ATCC Accession Number HTB-22) and MDA-MB-231(ATCC Accession Number HTB-26). A candidate modulator may be tested bytransfecting the cells with a polynucleotide encoding the candidate andevaluating the effect of expression of the polynucleotide on SPL levels.Alternatively, the cells may be contacted with a candidate modulator,typically in an amount ranging from about 10 μnM to about 10 mM. Acandidate that results in a statistically significant change in thelevel of SPL mRNA and/or protein is a modulating agent.

[0076] Alternatively, or in addition, a candidate modulating agent maybe tested for the ability to inhibit or increase SPL activity, using anin vitro assay as described herein (see Van Veldhoven and Mannaerts, J.Biol. Chem. 266:12502-07, 1991) that detects the degradation of labeledsubstrate (i.e., sphingosine-1-phosphate, or a derivative thereof).Briefly, a solution (e.g., a cellular extract) containing an SPLpolypeptide (e.g., 10 nM to about 10 mM) may be incubated with acandidate modulating agent (typically 1 nM to 10 mM, preferably 10 nM to1 mM) and a substrate (e.g., 40 μM) at 37° C. for 1 hour in the presenceof, for example, 50 mM sucrose, 100 mM K-phosphate buffer pH 7.4, 25 mMNaF, 0.1% (w/v) Triton X-100, 0.5 mM EDTA, 2 mM DTT, 0.25 mM pyridoxalphosphate. Reactions may then be terminated and analyzed by thin-layerchromatography to detect the formation of labeled fatty aldehydes andfurther metabolites. A modulating agent (e.g., an antibody) thatincreases SPL activity results in a statistically significant increasein the degradation of sphingosine-1-phosphate, relative to the level ofdegradation in the absence of modulating agent. Such modulating agentsmay be used to increase SPL activity in a cell culture or a mammal, asdescribed below.

[0077] A modulating agent may additionally comprise, or may beassociated with, a targeting component that serves to direct the agentto a desired tissue or cell type. As used herein, a “targetingcomponent” may be any substance (such as a compound or cell) that, whenlinked to a compound enhances the transport of the compound to a targettissue, thereby increasing the local concentration of the compound.Targeting components include antibodies or fragments thereof, receptors,ligands and other molecules that bind to cells of, or in the vicinityof, the target tissue. Known targeting components include hormones,antibodies against cell surface antigens, lectins, adhesion molecules,tumor cell surface binding ligands, steroids, cholesterol, lymphokines,fibrinolytic enzymes and other drugs and proteins that bind to a desiredtarget site. In particular, anti-tumor antibodies and compounds thatbind to an estrogen receptor may serve as targeting components. Anantibody employed in the present invention may be an intact (whole)molecule, a fragment thereof, or a functional equivalent thereof.Examples of antibody fragments are F(ab′)2, -Fab′, Fab and F[v]fragments, which may be produced by conventional methods or by geneticor protein engineering. Linkage may be via any suitable covalent bondusing standard techniques that are well known in the art. Such linkageis generally covalent and may be achieved by, for example, directcondensation or other reactions, or by way of bi- or multi-functionallinkers.

[0078] For in vivo use, a modulating agent as described herein isgenerally incorporated into a pharmaceutical composition prior toadministration. A pharmaceutical composition comprises one or moremodulating agents in combination with a physiologically acceptablecarrier. To prepare a pharmaceutical composition, an effective amount ofone or more modulating agents is mixed with any pharmaceuticalcarrier(s) known to those skilled in the art to be suitable for theparticular mode of administration. A pharmaceutical carrier may beliquid, semi-liquid or solid. Solutions or suspensions used forparenteral, intradermal, subcutaneous or topical application mayinclude, for example, a sterile diluent (such as water), salinesolution, fixed oil, polyethylene glycol, glycerine, propylene glycol orother synthetic solvent; antimicrobial agents (such as benzyl alcoholand methyl parabens); antioxidants (such as ascorbic acid and sodiumbisulfite) and chelating agents (such as ethylenediaminetetraacetic acid(EDTA)); buffers (such as acetates, citrates and phosphates). Ifadministered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, polypropylene glycol and mixtures thereof. In addition, otherpharmaceutically active ingredients (including other anti-cancer agents)and/or suitable excipients such as salts, buffers and stabilizers may,but need not, be present within the composition.

[0079] A modulating agent may be prepared with carriers that protect itagainst rapid elimination from the body, such as time releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, polyorthoesters, polylactic acid and others known to those ofordinary skill in the art.

[0080] Administration may be achieved by a variety of different routes,including oral, parenteral, nasal, intravenous, intradermal,subcutaneous or topical. Preferred modes of administration depend uponthe nature of the condition to be treated or prevented. An amount that,following administration, inhibits, prevents or delays the progressionand/or metastasis of a cancer is considered effective. Preferably, theamount administered is sufficient to result in regression, as indicatedby 50% mass or by scan dimensions. The precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by testing thecompositions in model systems known in the art and extrapolatingtherefrom. Controlled clinical trials may also be performed. Dosages mayalso vary with the severity of the condition to be alleviated. Apharmaceutical composition is generally formulated and administered toexert a therapeutically useful effect while minimizing undesirable sideeffects. The composition may be administered one time, or may be dividedinto a number of smaller doses to be administered at intervals of time.For any particular subject, specific dosage regimens may be adjustedover time according to the individual need.

[0081] As an alternative to direct administration of a modulating agent,a polynucleotide encoding a modulating agent may be administered. Such apolynucleotide may be present in a pharmaceutical composition within anyof a variety of delivery systems known to those of ordinary skill in theart, including nucleic acid, bacterial and viral expression systems, andcolloidal dispersion systems such as liposomes. Appropriate nucleic acidexpression systems contain the necessary DNA sequences for expression inthe patient (such as a suitable promoter and terminating signal, asdescribed above). The DNA may also be “naked,” as described, forexample, in Ulmer et al., Science 259:1745-49, 1993.

[0082] Various viral vectors that can be used to introduce a nucleicacid sequence into the targeted patient's cells include, but are notlimited to, vaccinia or other pox virus, herpes virus, retrovirus, oradenovirus. Techniques for incorporating DNA into such vectors are wellknown to those of ordinary skill in the art. Another delivery system forpolynucleotides is a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preparation and use ofliposomes is well known to those of ordinary skill in the art.

[0083] Within certain aspects of the present invention, one or moremodulating agents may be used to modulate SPL expression and/or activityin vitro, in a cell or in a mammal. In vitro, an SPL polypeptide may becontacted with a modulating agent that increases or decreases SPLactivity (e.g., certain antibodies). For use within a cell or a mammal,such modulation may be achieved by contacting a target cell with aneffective amount of a modulating agent, as described herein.Administration to a mammal may generally be achieved as described above.

[0084] As noted above, increase of SPL expression and/or activityprovides a method for inhibiting the growth (i.e., proliferation) of acancer cell, either in culture or in a mammal afflicted with cancer. Invivo, such increase may also be used to inhibit cancer development,progression and/or metastasis. Accordingly, one or more modulatingagents as provided herein may be administered as described above to amammal in need of anti-cancer therapy. Patients that may benefit fromadministration of a modulating agent are those afflicted with cancer.Such patients may be identified based on standard criteria that are wellknown in the art. Within preferred embodiments, a patient is afflictedwith breast cancer, as identified based on tissue biopsy and microscopicevaluation, using techniques well known in the art. In particular,patients whose tumor cells contain a tissue-specific deletion and/oralteration within an endogenous SPL gene may benefit from administrationof a modulating agent, as provided herein.

[0085] Within other aspects, the present invention provides methods andkits for diagnosing cancer and/or identifying individuals with a riskfor metastasis that is higher or lower than average. It has been found,within the context of the present invention, that certain human tumorcells contain an altered SPL gene. In particular, certain brain tumorcells contain a deletion of amino acid residues 354 to 433 of the humanSPL sequence set forth in SEQ ID NO:8 (cDNA and amino acid sequence ofthe SPL containing the deletion are set forth in SEQ ID NOs:9 and 10,respectively). Specific alterations present in other tumor cells, suchas breast tumor cells, may be readily identified using standardtechniques, such as PCR. Alterations that may be associated with apaticular tumor include amino acid deletions, insertions, substitutionsand combinations thereof Methods in which the presence or absence ofsuch an alteration is determined may generally be used to detect cancerand to evaluate the prognosis for a patient known to be afflicted withcancer.

[0086] To detect an altered SPL gene, any of a variety of well-knowntechniques may be used including, but not limited to, PCR andhybridization techniques. Any sample that may contain cancerous cellsmay be assayed. In general, suitable samples are tumor biopsies. Withina preferred embodiment, a sample is a breast tumor biopsy.

[0087] Kits for diagnosing or evaluating the prognosis of a cancergenerally comprise reagents for use in the particular assay to beemployed. In general, a kit of the present invention comprises one ormore containers enclosing elements, such as primers, probes, reagents orbuffers, to be used in an assay. For example, a kit may contain one ormore polynucleotide primers or probes comprising at least 15 nucleotidescomplementary to a polynucleotide encoding SPL. In certain embodiments,the primers or probes comprise at least 10, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides, and preferably atleast 150 or 200 nucleotides, complementary to an SPL mRNA or to apolynucleotide encoding SPL. Such probe(s) may be used to detect analtered SPL gene by hybridization. For example, a kit may contain oneprobe that hybridizes to a region of an SPL gene that is not generallyaltered in tumors (a control) and a second probe that hybridizes to aregion commonly deleted in breast cancer. A sample that contains mRNAthat hybridizes to the first probe, and not to the second (usingstandard techniques) contains an altered SPL gene. Suitable controlprobes include probes that hybridize to a portion of the SPL geneoutside of the commonly deleted region encoding amino acid resides 354to 433; suitable probes for an altered region include probes thathybridize to a portion of the SPL gene that encodes amino acid residues354 to 433. Alternatively, a kit may comprise one or more primers forPCR analyses, which may be readily designed based upon the sequencesprovided herein by those of ordinary skill in the art. Optionally, a kitmay further comprise one or more solutions, compounds or detectionreagents for use within an assay as described above.

[0088] In a related aspect of the present invention, kits for detectingSPL are provided. Such kits may be designed for detecting the level ofSPL or nucleic acid encoding SPL within a sample, or may detect thelevel of SPL activity as described herein. A kit for detecting the levelof SPL, or nucleic acid encoding SPL, typically contains a reagent thatbinds to the SPL protein, DNA or RNA. To detect nucleic acid encodingSPL, the reagent may be a nucleic acid probe or a PCR primer. To detectSPL protein, the reagent is typically an antibody. The kit may alsocontain a reporter group suitable for direct or indirect detection ofthe reagent as described above.

[0089] Within further aspects, the present invention provides transgenicmammals in which SPL activity is reduced, compared to a wild-typeanimal. Such animals may contain an alteration, insertion or deletion inan endogenous SPL gene, or may contain DNA encoding a modulating agentthat modulates expression or activity of an SPL gene. In certainaspects, such animals may contain DNA encoding a modulating agent thatincreases expression or activity of an SPL gene. Transgenic animals maybe generated using techniques that are known to those of ordinary skillin the art. For example, a transgenic animal containing an insertion ordeletion in the coding region for the SPL gene may be generated fromembryonic stem cells, using standard techniques. Such stem cells may begenerated by first identifying the full genomic sequence of the geneencoding the SPL, and then creating an insertion or deletion in thecoding region in embryonic stem cells. Alternatively, appropriategenetically altered embryonic stem cells may be identified from a bank.Using the altered stem cells, hybrid animals may be generated with onenormal SPL gene and one marked, abnormal gene. These hybrids may bemated, and homozygous progeny identified.

[0090] Transgenic aminals may be used for a variety of purposes, whichwill be apparent to those of ordinary skill in the art. For example,such animals may be used to prepare cell lines from different tissues,using well known techniques. Such cell lines may be used, for example,to evaluate the effect of the alteration, and to test various candidatemodulators.

[0091] The invention further provides Drosophila melanogaster animalmodels that exhibit a flightless phenotype, where the phenotype resultsfrom the disruption of an endogenous SPL gene as described in greaterdetail below. By flightless phenotype is meant that the subjectnon-mammalian animal models spontaneously develop a reduced number ofmuscle fibers comprising the dorsal longitudinal muscles (DLM) and havecompensatory hypertrophy in the remaining fibers. In certain aspects,the non-mammalian animal model of the present invention may alsodemonstrate abnormal developmental patterning of thoracic muscles of theT2 segment. In a preferred embodiment, the above phenotypes result in aninability to fly. The subject non-mammalian animal models, within apreferred embodiment, demonstrate altered activity of the endogenousSPL. In a particularly illustrative embodiment, said D. melanogasteranimal models have decreased activity of endogenous SPL.

[0092] Within further aspects, the present invention provides mutantstrains of Drosophila melanogaster. In a preferred embodiment, thestrain contains a mutation in the SPL gene. In a further embodiment ofthe present invention the D. melanogaster strain are heterozygous for aP-element transposon which sits in the coding region of the geneencoding the SPL protein set forth in SEQ ID NO:16. In a preferredembodiment, the flies are homozygous insertional mutants in the codingregion of the gene encoding the SPL protein set forth in SEQ ID NO:16.In yet a further embodiment of the present invention, the homozygousmutant strain of fly has a flightless phenotype. In certain embodiments,the mutant flies have a reduced number of muscle fibers comprising thedorsal longitudinal muscles and have compensatory hypertrophy in theremaining fibers. In certain aspects, the mutant flies of the presentinvention may also demonstrate abnormal developmental patterning ofthoracic muscles of the T2 segment.

[0093] Flies heterozygous for a P-element transposon which sits in thecoding region of the SPL gene or genes and and disrupts production ofSPL proteins may be obtained from the Drosophila Genome Project.Homozygous insertional mutants can be made, using techniques known inthe art, by genetically crossing and evaluating progeny for the presenceof homozygous insertional mutants (based on presence of rosy eye color,encoded by a recessive marker carried on the P-element). Expression ofthe SPL gene can be evaluated using any number of assays known to theskilled artisan, for example, by Northern blot analysis. To determinethe SPL function of each genotype, +/+, +/− and −/− flies may behomogenized using standard techniques and whole extracts can be assayedfor SPL activity using assays as described herein. The transposon can bemobilized by crossing SPL mutant flies with flies carrying an activelytranscribed transposase gene, which should cause the P-element to beexcised in the chromosomes of both somatic cells and in the germline.Germline transposon loss is heritable and can be identified in progenyby virtue of eye color. Progeny which lost both the transposase gene andthe P-element can then be isolated and the restored SPL allele can behomozygosed.

[0094] Mutations in Drosophila melanogaster as described herein whichpermanently block expression of a functional protein can be created inseveral ways, such as with P-element transposon insertions or chemicalor radiation induced mutagenesis. Exemplary strains of mutant flies areavailable through the Drosophila Genome Project, at the University ofCalifornia at Berkeley (Adams, M. et al 2000. The genome sequence ofDrosophila melanogaster. Science. 287:2185-2195.). Alternatively,insertional mutant of interest may be obtained by using local hopstrategies essentially as described in Tower, J. et al (Tower, J., etal. 1993. Preferential transposition of Drosophila P elements to nearbychromosomal sites. Genetics. 133:347-359.), hereby incorporated byreference in its entirety. Transposons can be mobilized by crossing in atransposase gene, followed by crossing the transposase back out(reintroducing genetic stability). Mutant flies can be identified usingtechniques know to those of skill in the art. For example, mutant fliescan be identified by probing Southern blots prepared from extracts fromflies generated in the screen using the target gene as probe.Subsequently, crosses can be performed to introduce a mutant allele ofinterest, (e.g. SPL) and generate homozygosity at both mutant alleles(e.g. SPL and new transposon integration sites). Mutants can be screenedfor a phenotype of interest, for example the ability to restore flightto an SPL mutant when the mutated allele is homozygous (predicting arecessive phenotype).

[0095] In one aspect of the present invention, fly genetic manipulationmay entail mating or “crossing” of flies and selection for or againstprogeny expressing various phenotypic markers. Exemplary techniques forfly genetic manipulation of the present invention are know in the artand are described, for example in, Ashburner, M., and J. Roote. 2000.Laboratory culture of Drosophila. In Drosophila Protocols. W. Sullivan,M. Ashburner, and R. Hawley, editors. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. 585-600. Phenotypic markers may be usedto identify the inheritance of chromosomes, engineered transposableelements, or transposase genes used to facilitate their mobilization.Marker mutations affecting eye color, bristle shape, wing morphology andcuticle pigmentation, for example, may be employed in the crosses forthe mutant flies of the present invention. Within one aspect of thepresent invention, it may be desirable to select the individuals whichcontain a collection of markers indicating the desired genotype. Inanother aspect of the present invention, balancer chromosomes may beused to create the ability to identify recessive mutations present inthe heterozygous state. Balancer chromosomes may be employed to preventhomologous recombination during meiotic prophase in females. Thepresence of both dominant and recessive lethal markers allows one todetermine the presence or absence of the balancer chromosomes andsimultaneously to follow the homologous chromosomes, which maythemselves not contain a dominant marker. One particularly illustrativecross of the present invention is to eliminate the P-element insertionin the Drosophila SPL gene and establish phenotypic reversion, asdescribed herein in the Examples.

[0096] Selective markers to allow for selection of mutant flies isprovided for in the present invention. Exemplary selective markers ofthe present invention may comprise a wild type rosy (ry⁺) allele carriedon the transposon to allow for selection for or against the stabletransposon. Introduction of an active transposase is selected for bypresence of the dominant marker, Stubble (short bristle phenotype) inthe first cross, and is selected against to identify progeny which havelost the transposase, restoring genetic stability in the second cross.Other illustrative markers include Curly O (CyO) which is lethal whenpresent in two copies, allowing selection for heterozygotes containingthe CyO balancer and another allele of interest originally containingthe transposon (e.g., SPL). By selecting against rosy eye color, progenyin which the transposon has been excised from the locus of interest,e.g., SPL, can be identified. Expansion of this “reverted” allele in thepopulation can be achieved in the third cross, and the desired allelecan be homozygosed in the final cross, to determine whether restorationof the intact allele of interest, for example SPL, is associated with adesired phenotype of interest, such as restoration of flight.

[0097] In another aspect of the present invention, transgenic flies canbe created using P-elements to overexpress or misexpress proteins ofinterest, such as SPL. In one embodiment of the invention, GAL4-mediatedectopic gene expression is employed, essentially as described (vanRoessel, P., and A. Brand. 2000. GAL4-mediated ectopic gene expressionin Drosophila. In Drosophila Protocols. W. Sullivan, M. Ashburner, andR. Hawley, editors. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 439-448.). The GAL4 protein is a yeast transcription factorcapable of activating transcription of Drosophila genes which have beenengineered to contain upstream sequences recognized by the GAL4 protein.Various mutants can be created with a gene of interest expressed inspecific tissue distributions, a construct containing the gene ofinterest (reporter) under regulation of a GAL4 containing promoter isintroduced into embryos, and a genetic marker allows identification ofprogeny containing this construct. Illustrative GAL4 containingpromoters include, but are not limited to, pUAS. The use of embryos of astrain containing an active P-transposase increases the efficiency oftransgene integration, although many of the embryos die. These progenycan then be crossed to various available lines containing GAL4transgenes (driver) expressed under control of tissue-specificpromoters. In one aspect of the present invention, GAL4 driverconstructs which allow expression during embryogenesis are used.

[0098] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 Isolation and Characterization of SPL cDNA from Yeast

[0099] This Example illustrates the preparation of an S. cerevisiae cDNAmolecule encoding an endogenous SPL polypeptide.

[0100] Wild-type yeast cells (SGP3 (Garrett and Broach, Genes and Dev.3:1336-1348, 1989); leu2-3,112 trp1 ura3-52 his3 ade8 ras1::HIS3) weretransformed with a yeast genomic library carried on the pRS202 high-copyshuttle vector (Sikorski and Heiter, Genetics 122:19-27, 1989)containing a selectable nutritional marker (URA3). pRS202 is a modifiedversion of the pRS306 vector, into which a 2 micron plasmid piece wasinserted. Inserts from this library are approximately 6-8 kb in length.Wild type yeast were transformed with the high copy library as describedby Ito et al., J. Bact. 153:163-68, 1983, selected for uracilprototrophy (i.e., the ability to grow on medium lacking uracil), andtransformants were pooled and replated at a concentration of 10⁶ cellsper plate onto 1 mM D-erythro-sphingosine plates.

[0101] Six transformants which grew large colonies on 1 mMD-erythro-sphingosine plates were grown in selective medium, and controlSGP3 colonies were grown in minimal medium, at 30° C. until saturated.Absorbance at 660 nm was used to correct for small variations in cellconcentration between cultures. Serial dilutions were performed, andcells were template-inoculated onto 1 mM D-erythro-sphingosine platesand incubated at 30° C. for 48 hours.

[0102] The most highly represented insert, 13-1, was subdloned andsequenced, and named BST1 (bestower of ghingosine tolerance; GenBankaccession number U51031; Saccharomyces cerevisiae genome databaseaccession number YDR294C). The BST1 nucleotide sequence encodes apreviously unknown predicted protein of 65,523 kilodaltons and 589 aminoacids in length. This sequence is 23% identical to gadA and gadB, twonearly identical E. coli genes encoding glutamate decarboxylase (GAD), apyridoxal-5′-phosphate-dependent enzyme which catalyzes synthesis of theneurotransmitter γ-amino butyric acid. BST1 has been localized to S.cerevisiae chromosome 4. The DNA sequence of BST1 is provided in SEQ IDNO:1, which encodes the amino acid sequence set forth in SEQ ID NO:2.

[0103] To explore the function of BST1, a deletion strain was createdthrough homologous recombination using a NEO selectable marker (Wach etal., Yeast 10:1793-1808, 1994). Genomic BST1 was replaced with kanMX(Wach et al., Yeast 10:1793-1808, 1994), which confers resistance toG418. Disruption was confirmed using PCR amplification of genomic DNAfrom G418 resistant clones, using primers to genomic sequence just 5′and 3′ to the region replaced by the disruption. Deletion of BST1 andall subsequent biological studies were performed in both SGP3 and inJK93d (Hietman et al., Proc Natl. Acad. Sci. USA 88:1948-52, 1991);ura3-52 leu2-3,112 his4 trp1 rme1). Heterozygous diploids weresporulated, and spores segregated 2:2 for G418 resistance. Both G418resistant and sensitive progeny were viable, indicating that BST1 is notan essential gene.

[0104] Analysis of GAD activity in cytosolic extracts from wild type,BST1 overexpression and bst1Δ strains indicated that BST1 does notencode the S. cervisiae homologue of GAD. However, deletion of BST1 wasassociated with severe sensitivity to D-erythro-sphingosine.Concentrations as low as 10 μM sphingosine completely inhibited growthof bst1Δ strains but had no effect on the viability of wild type cells.In comparison to the control strain, the bst1Δ strain also demonstratedgreater sensitivity to 100 μM phytosphingosine, the long chain baseendogenous to S. cerevisia. No difference between the growth of wildtype and BST1 overexpression strains on phytosphingosine, which is onlyminimally toxic to wild type cells at this concentration, was observed.

[0105] To determine whether differences in sphingosine uptake ormetabolism were responsible for these sensitivity differences, BST1 wildtype, overexpression and bst1Δ strains were exposed to [C3-³H] labeledsphingosine (American Radiolabeled Chemical, Inc., St. Louis, Mo.),washed in sterile water and subjected to Bligh-Dyer extractions (Blighand Dyer, Can. J. Buichem. Physiol. 37:911-17, 1959). There were nomajor differences in sphingosine recovery among the three strains.However, the aqueous phase from the bst1Δ strain contained a ten-foldincrease in radioactivity over that of control and BST1 overexpressionstrains. Thin layer chromatography (TLC) analysis of the lipid fractionsin butanol:acetic acid:water (3:1:1) revealed a sphingosine band whichappeared equivalent in each strain.

[0106] Radioactive sphingosine-1-phosphate (S-1-P) was also observed inthe extracts from the bst1Δ strain, but not in the wild type or BST1overexpression strains. This compound accumulated rapidly, reaching aplateau by 60 minutes. Three separate TLC conditions were used toconfirm the presence of S-1-P. These conditions, along with theresulting RF values, are shown below: butanol:water:acetic acid (3:1:1).47 chloroform:methanol:water (60:35:8) .22chloroform:methanol:water:acetic acid (30:30:2:5) .33

[0107] Hyperaccumulation of S-1-P and hypersensitivity toD-erythro-sphingosine suggeset a failure to metabolize S-1-P, indicatingthat BST1 is a yeast SPL. To confirm this identification, lyase activityin BST1 wild type, overexpression and deletion strains were evaluated asdescribed by Veldhoven and Mannaerts, J. Biol. Chem. 266:12502-07, 1991,using unlabeled D-erythro-dihydrosphingosine-1-phosphate (Biomol,Plymouth Meeting, Pa.) and D-erythro-dihydrosphingosine[4,5-³H]1-phosphate (American Radiolabeled Chemicals, Inc., St. Louis,Mo.). Specific activity was 100 mCi/mmol. SPL activity was found tocorrelate with BST1 expression, confirming BST1 to be the yeasthomologue of sphingosine-1-phosphate lyase.

[0108] These results indication that BST1 is a yeast SPL, and that SPLcatalyzes a rate-limiting step in sphingolipid catabolism. Regulation ofSPL activity may therefore result in regulation of intracellular S-1-Plevels.

Example 2 Isolation and Characterization of SPL cDNA from C. elegans andMouse

[0109] This Example illustrates the identification of endogenous SPLcDNAs from C. elegans and Mus musculus.

[0110] Comparison of the yeast BST1 sequence to sequences within theGenBank database identified a full length gene from C. elegans that wasidentified during the systematic sequencing of the C. elegans genome.This cDNA sequence is set forth in SEQ ID NO:3 and was found to encodeSPL, the sequence set forth in SEQ ID NO:4. This and other DNA homologysearches described hereinwere performed via the National Center forBiotechnology Information website using BLAST search program.

[0111] Using both S. cerevisiae and C. elegans SPL sequences to searchthe EST database, an expressed sequence tag from early embryonic cellsof the mouse (day 8 embryo, strain C57BL/6J) was identified. The cDNAclone containing this putative mouse SPL was purchased from GenomeSystems, Inc (St. Louis, Mo.). Completion of the full length cDNAsequence revealed an 1707 bp open reading frame. This mouse sequenceshowed significant homology to BST1 and to other pyridoxalphosphate-binding enzymes such as glutamate decarboxylase, with greatestconservation surrounding the predicted pyridoxal phosphate-bindinglysine. Since the two genes encoding mouse glutamate decarboxylase havebeen identified previously, and the identified sequence was unique andhad no known function, it was a likely candidate mouse SPL gene.

[0112] To confirm the SPL activity of the mouse gene, a two step processwas undertaken. First, the sequence was cloned into the high-copy yeastexpression vector, pYES2 (Invitrogen, Inc., Carlsbad, Calif.), in whichthe gene of interest is placed under control of the yeast GAL promoterand is, therefore, transcriptionally activated by galactose andrepressed by glucose. pYES2 also contains the URA3 gene (which providestransformants the ability to grow in media without uracil) and anampicillin resistance marker and origin of replication functional in E.coli.

[0113] The expression vector containing the full-length mouse SPL genewas then introduced into the yeast bst1Δ strain whichn as noted above,is extremely sensitive to D-erythro-sphingosine, as a result ofmetabolism of sphingosine to S-1-P. S-1-P cannot be further degraded inthe absence of SPL activity and overaccumulates, causing growthinhibition. Transformation was performed using the lithium acetatemethod (Ito et al., J. Bact. 153:163-68, 1983). Transformants were grownon medium containing 20 g/L galactose and selected for uracilprototrophy.

[0114] Transformants were then evaluated for sphingosine resistance.Strains of interest were grown to saturation in liquid culture for 2-3days. They were then resuspended in minimal medium, placed in the firstrow of a 96-well plate and diluted serially from 1:2 to 1:4000 acrossthe plate. The cultures were then template inoculated onto a controlplate (YPD) and a plate containing minimal synthetic media supplementedwith 50 μM D-erythro-sphingosine (Sigma Chemical Co., St. Louis, Mo.)and 0.0015% NP40 (Sigma Chemical Co.). At this concentration of NP40, noeffects on cell viability were observed. Plates were incubated at 30° C.for two days and assessed visually for differences in growth.Transformants containing the mouse SPL gene were resistant tosphingosine present in galactose-containing plates. A strain transformedwith vector alone remained sensitive to sphingosine. Therefore, themouse SPL gene was capable of reversing the sphingosine-sensitivephenotype of a yeast bst1Δ strain.

[0115] In order to determine whether the mouse SPL gene was able torestore biochemical SPL activity to the bst1Δ strain, the untransformedbst1Δ strain, and the bst1Δ strain transformed with pYES2 containingeither BST1 or the putative mouse SPL gene were grown to exponentialphase (A₆₀₀=1.0) in either minimal (JS16) or uracil medium containinggalactose as a carbon source. Whole cell extracts were prepared fromeach strain as described above, adjusted for protein concentration, andevaluated for sphingosine phosphate lyase activity as described above,using ³H-dihydrosphingosine-1-phosphate (American RadiolabeledChemicals, Inc., St. Louis, Mo.). Qualitative analysis of product wasperformed by autoradiography. Quantitative measurement was performed byscraping TLC plates and determining radioactivity present using astandard scintillation counter.

[0116] The results of the sphingosine phosphate lyase assays shownedthat expression of both the yeast and mouse sequences restored SPLactivity to the bst1Δ strain, whereas vector alone had no effect,confirming the identity of the mouse sequence as SPL.

[0117] To determine whether the expression of the mouse SPL transcriptcoincided with previously reported tissue-specific SPL activity in themouse, total RNA was obtained from a variety of mouse tissues and probedwith the complete mouse SPL cDNA sequence. Northern analysis wasperformed as described by Thomas, Proc. Natl. Acad. Sci. USA 77:5201,1980, using a full length mouse SPL cDNA probe labeled by randomlabeling technique (Cobianchi and Wilson, Meth. Enzymol. 152:94-110,1987). This analysis revealed a pattern of expression consistent withthe known SPL activity in various mouse tissues, providing furtherconfirmation that this sequence encodes mouse SPL.

Example 3 Isolation and Characterization of Human SPL cDNA

[0118] This Example illustrates the identification of an endogenoushuman cDNA.

[0119] An EST database was searched using the mouse SPL sequencedescribed herein. Two distinct EST sequences having strong homology tothe mouse sequence were identified from human sources. One of thesesequences corresponded to the C-terminus, and the other corresponded tothe N-terminus. Primers were designed based on these sequences, and aDNA fragment was amplified by PCR from a human expression library madefrom human glioblastoma multiforme tissue RNA. The fragment wassequenced and was shown to contain a deletion, so the primers were usedto amplify the gene from human fibroblast RNA. This gene has thesequence provided in SEQ ID NO:7 and encodes the polypeptide sequenceprovided in SEQ ID NO:8. The cDNA and amino acid sequences of the SPLcontaining the deletion are set forth in SEQ ID NOs:9 and 10,respectively.

Example 4 Isolation and Characterization of C. Elegans SPL cDNA

[0120] This Example illustrates the identification of a cDNA moleculeencoding a primary C. elegans sphingosine phosphate lyase.

[0121] The human SPL cDNA sequence was used to screen the ACEdb C.elegans genome database. A potential C. elegans open reading frame ofunknown function present on YAC Y66H1B showed substantial (40%) homologyto yeast, human and mouse SPL cDNA sequences. To clone this sequence, acoupled reverse transcriptase/polymerase chain reaction was performedusing the Access RT-PCR system (see below). Template was C. eleganstotal RNA, and primers were: 5′-GAGGAATTCATGGATTCGGTTAAGCACACAACCG-3′5′-AGCCTCGAGTTAATTAGAAGTTGAAGGTGGAGC-3′

[0122] This resulted in a DNA fragment cSPL2, which was ligated into theyeast expression vector pYES2, obtained from Invitrogen. Inc. (Carlsbad,Calif.). Genes expressed using this system are regulated under thecontrol of the GAL promoter, which allows expression in the presence ofgalactose and not in the presence of glucose. The nucleotide sequence ofcSPL2 is set forth in SEQ ID NO:12, with the encoded amino acid sequenceset forth in SEQ ID NO:11

[0123] cSPL2 was further analyzed for its ability to complement thesphingosine sensitive phenotype of a yeast dpl1 mutant, the previouslydescribed yeast strain JS16 which contains a large deletion in DPL1, theS. cerevisiae sphingosine phosphate lyase gene (Zhou and Saba, BiochemBiophys Res Commun 242:502-507, 1998). Transformation of JS16 with pYES2or the C. elegans SPL-pYES2 construct was performed by the lithiumacetate method (Ito et al., J. Bact. 153:163-168, 1983). Transformantswere selected for uracil prototrophy and evaluated for a sphingosineresistance using the dilutional assay described by Zhou and Saba,Biochem Biophys Res Commun 242:502-507, 1998. Cells were grown inminimal or uracil³¹ media containing either 20 g glucose or galactoseper liter, as indicated. D-erythro-sphingosine and NP40 were obtainedfrom Sigma Chemical Company (St. Louis, Mo.).

[0124] The results demonstrate that cSPL2 convincingly complemented theyeast mutant, restoring enzyme activity. In each plate, yeast were grownto saturation in overnight liquid cultures, spun down, resuspended in200 microliters of water and dispensed into the first (left-most) wellof each horizontal row. Yeast were then further diluted into sterilewater, so the second well was 1:2, third well was 1:4, fourth well was1:40, fifth was 1:400 and sixth was 1:4000 dilution from the original onthe left. The toxicity of sphingosine is cell number dependent, becauseit disperses itself in cell membranes. Therefore, the concentration ofsphingosine in the plate is not the only thing affecting toxicity, andthese dilutional assays show differences in tolerance/sensitivity. So, astrain which can grow in the sixth row is about 4,000 times moreresistant to sphingosine than one which can grow only in the first row.

[0125] The mutant yeast strain containing cSPL2 also demonstratedsubstantial SPL activity. The sphingosine phosphate lyase assay usedwhole cell extracts of yeast containing either pYES2 vector alone or(cSPL2) C. elegans SPL-pYES2. Extracts were prepared as described bySaba et al., J Biol Chem 272:26087, 1997. SPL activity was determinedessentially as described, using ³H-dihydrosphingosine-1-phosphatesubstrate (see Zhou and Saba, Biochem Biophys Res Commun 242:502-507,1998). Substrate for SPL assay (³H-dihydrosphingosine-1-phosphate) wasobtained from American Radiolabeled Chemicals, Inc. (St. Louis, Mo.).Access RT-PCR system was obtained from Promega Corp. (Madison, Wis.).

[0126] Enzyme activity in (cSPL2) C. elegans SPL-pYES2 was appreciablygreater than that of the vector control. These results indicate thatcSPL2 encodes the primary C. elegans SPL.

Example 5 Developmental Defects Induced by RNA Interference in C.elegans

[0127] In order to determine the effect of blocking cSPL2 expression onthe development of C. elegans, RNA interference studies were undertaken.The cSPL2 cDNA was cloned into pBluescript such that the insert wasflanked by the T3 and T7 promoter regions. RNA complementary to eachstrand was synthesized from these promoters using an in vitrotranscription kit (Promega, Madison, Wis.). The two strands wereannealed to make double stranded RNA (dsRNA) and injected into thedistal gonads of 12 wild-type (N2 Bristol) young adult C. eleganshermaphrodites. As controls, uninjected hermaphrodites as well ashermaphrodites injected with a dsRNA that does not produce a visiblephenotype were handled in parallel. Eight hours after injection, eachhermaphrodite was transferred to a fresh culture plate and 12 hourcohorts of F1 progeny were established. Progeny were observed daily witha dissecting microscope until most animals reached adulthood and theculture plates became too crowded with F2 progeny. Compared to controlF1s, animals inheriting cSPL-2 dsRNA developed slowly, moved sluggishly,were thin and pale, and did not pump food actively. These animals reachadulthood approximately 24 hours later than controls. Adulthermaphrodites that inherited cSPL-2 dsRNA were markedly different fromcontrols especially in the gonad and uterus. Control animals hadabundant nuclei in the distal gonad and a row of developing oocytes inthe proximal gonad. Affected hermaphrodites had poorly developed distalgonads with fewer nuclei. Control adults had embryos of progressivestages of development in the uterus, whereas the number of developingoocytes in the proximal gonad of affected hermaphrodites was reduced.The embryos in the uterus of affected progeny were also abnormal. Thosenear the vulva were at late developmental stages indicating a defect inegg laying. There was not a uniform progression of developmental stagesin adjacent embryos suggesting a defect in ovulation or development, andsome of the embryos showed abnormal patterns of cell division. Insummary, inhibition of C. elegans SPL expression through the use of RNAinterference leads to poor feeding, developmental abnormalities andimpaired fertility in the progeny. These results suggest that SPL is anessential gene in C. elegans.

Example 6 Isolation and Characterization of SPL cDNA from Drosophilamelanogaster

[0128] In order to seek out the Drosophila melanogaster SPL cDNA andgenomic sequence, the D. melanogaster genomic database was searched forsequences which demonstrated significant homology to human SPL cDNA.This led to identification of two full-length cDNA clones (LP04413 andGH3783) which were confirmed by sequence and restriction analysis. Thetwo clones are predicted based on alternative 5′ exon usage and may beexpressed in different subcellular locations. The predicted Drosophilamelanogaster SPL is located on the right arm of chromosome II, position53F8-12. The cDNA sequence for Drosophila melanogaster SPL is set forthin SEQ ID NO:15 and encodes the SPL protein set forth in SEQ ID NO:16.The Drosophila SPL predicted protein sequence set forth in SEQ ID NO:16is 49%, 49% and 43% identical to human, mouse and yeast SPL proteinsequences, respectively.

[0129] In order to evaluate whether these clones contained a functionalSPL gene, they were recloned into the yeast expression vector, pYES2,and this construct was transformed into a dpl1Δ strain. Expression ofclones containing the potential Drosophila melanogaster SPL fullycomplement the dpl1Δ strain's sensitivity to 50 μMD-erythro-sphingosine. Further, whole cell extracts of dpl1 strainscontaining either pYES2-LP04413 or pYES2-GH3783 demonstrate restorationof SPL enzyme activity to wild type levels or greater, although not ashigh as a DPL1 overexpressing strain (DPL OE).

Example 7 Generation and Characterization of SPL Transposon Mutant D.melanogaster

[0130] Flies heterozygous for a P-element transposon which sits in thecoding region of both of the above transcripts described in Example 6and presumably disrupts both SPL proteins were obtained from theDrosophila Genome Project. These flies were genetically crossed usingtechniques well known to ordinarily skilled artisans, and progeny wereevaluated for the presence of homozygous insertional mutants (based onpresence of rosy eye color, encoded by a recessive marker carried on theP-element). Northern blot analysis from wild type and SPL insertionalmutant flies indicated that no SPL gene expression occured in thelatter.

[0131] To determine the SPL function of each genotype, +/+, +/− and −/−flies were homogenized and whole extracts assayed for SPL activity. Itwas observed that SPL genotype corresponded with SPL activity with +/+ >+/− > −/−. Initial evaluation of homozygous mutants indicated that adultSPL mutants were flightless, suggesting a potential defect in eithermuscle development or energetics of the adult fly. Flight analysis wascarried out essentially as described (Vigoreaux, J., J. Saide, K.Valgeirdottir, and M. Pardue. 1993. Flightin, a novel myofibrillarprotein of Drosophila stretch-activated muscles. J Cell Biol.121:587-598) by determining the percentage of flies that were flightlessor exhibited downward, upward, or lateral flight capabilities in controlCanton-S flies as compared to mutant flies.

[0132] The transposon was mobilized by crossing SPL mutant flies withflies carrying an actively transcribed transposase gene, which causedthe P-element to be excised in the chromosomes of both somatic cells andin the germline. Germline transposon loss is heritable and wasidentified in progeny by virtue of eye color. Progeny which lost boththe transposase gene and the P-element were then isolated and therestored SPL allele was homozygosed. Progeny which had lost theP-element at the SPL locus demonstrated restoration of flight,indicating that the phenotype correlated with the P-element insertionalmutation. To determine the etiology of the flightlessness of −/− flies,flies were sectioned through the thoracic region and indirect flightmuscles were evaluated by both light and electron microscopy. Thesestudies revealed a reduced number of muscle fibers comprising the dorsallongitudinal muscles with evidence of what appears to be compensatoryhypertrophy in the fibers which remained. Electron microscopy revealedno ultrastructural defects in the myocytes which remained.

[0133] In order to determine whether the loss of SPL expression was dueto excess accumulation of S-1-P in the developing adult fly, we salvagedthe developing flight muscles of homozygous SPL mutant progeny by addingD,L-threo-dihydrosphingosine, an inhibitor of mammalian sphingosinekinase, to the growth media. A significant proportion of homozygous SPLmutant progeny demonstrated restoration of flight when grown on mediasupplemented with D,L-threo-dihydrosphingosine.

[0134] Northern analysis was performed to investigate SPL expressionthroughout development. These studies indicated that SPL expressionbegins at 8-12 hours of embryonic development and remains detectiblethroughout larval stages and pupation.

[0135] Therefore, the Drosophila melanogaster model described herein canbe used to identify pharmacologic suppressors of SPL mutant flies'inability to fly. Drugs which alter SPL activity or expression may beeffective treatment for at least some kinds of cancer. Therefore, thefact that a fruitfly SPL null mutant containing a P-element insertionwithin the SPL coding region is flightless provides an excellent modelin which to screen and identify compounds that modulate SPL activity.Thus, other chemicals created through rational drug design approachescan be screened using this method. The Drosophila melanogaster modeldescribed herein can thus be used to screen an array of rationallydesigned chemicals with homology to sphingolipids for their ability torestore flight to SPL mutant progeny. Candidate drugs identified usingthis method can then be further evaluated in an in vitro yeast screen.

Example 8 Further Characterization of Developmental Expression Patternsof SPL in SPL Transposon Mutant D. melanogaster

[0136] Northern analysis is carried out and extended to include adultsamples, and blots are reprobed with SPL specific probes using thefollowing approaches. Once genes are confirmed to encode the predictedenzyme, DNA probes or riboprobes for SPL and S-1-P phosphatase arelabeled either radioactively or with digoxygenin. For Northern analysis,full-length probes are labeled by random priming with [α-³²P]dATP.Hybridization is carried out under standard conditions against an RNAblot prepared from total RNA of flies harvested at different stages ofdevelopment (embryos at hours 0-4, 4-8, 8-12, 12-24, larval instars1^(st), 2^(nd), 3^(rd), early and late pupal stages, and adults). For insitu hybridization purposes, ³H labeling is the most sensitive approach,and the very low energy of the beta particle emitted causes it to travelonly short distances through the radiographic emulsion, allowing preciselocalization for the probe. However, digoxygenin labeling provides theadvantage of being able to visualize hybridization with much higherspatial resolution because of the ability to directly visualize thetissue. Random primer labeling of DNA are performed with either tritiumor digoxygenin labeled nucleotides. In situ hybridization is performedas described in Blair, S. (Blair S., 2000. Imaginal discs. In DrosophilaProtocols. W. Sullivan, M. Ashburner, and R. Hawley, editors. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 159-175),hereby incorporated by reference in its entirety.

Example 9 Characterization of Sphingolipid Species in the Drosophilamelanogaster

[0137] Without being bound by theory, it is hypothesized that thephenotype of the SPL mutant Drosophila is caused by an abnormal level ofS-1-P during development. Further, without being bound by theory, it isthe inventors hypothesis that phosphorylated sphingoid base species areresponsible for regulating cell proliferation, migration and otherevents required for both tumor formation and normal developmentalprocesses in this model organism. Therefore, characterization ofsphingolipid species in Drosophila was determined.

[0138] Method: Wild type (Canton S) whole fly extracts were prepared bymechanical disruption. Lipids were isolated by two-phase extraction andderivatized with the fluorescent molecule o-pthalaldehyde essentially asdescribed in Caligan, et al. hereby incorporated by reference in itsentirety (Caligan, T. B., K. Peters, J. Ou, E. Wang, J. Saba, and A. H.Merrill, Jr. 2000. A high-performance liquid chromatographic method tomeasure sphingosine 1-phosphate and related compounds from sphingosinekinase assays and other biological samples. Analytical Biochemistry.281:36-44). Derivatized lipid extracts were separated by HPLC using aC₁₈ ODS column (LUNA 4.6×250 mm) and mobile phase MeOH/H₂O/1M TBAP82:17:0.9, pH 4.8. Standards included commercially available C₁₀, C₁₂,C₁₄, C₁₆, C₁₈ and C₂₀ sphingosines, as well as the phosphorylated formsof these standards, prepared by incubation of sphingosine standards withextract from a yeast strain which overexpresses the major yeastsphingosine kinase, LCB4.

[0139] Results: Drosophila extracts contained only sphingolipid specieswhich comigrated with C₁₄ sphingosine and C₁₄ sphingosine-1-phosphate(S-1-P) standards under the stated conditions. To verify the identity ofthe peaks in fly extracts which comigrated with C₁₄sphingosine andC₁₄S-1-P standards, extracts and standards were compared in fourdifferent mobile phase buffers. The peak identified as C₁₄ sphingosinecomigrated with the C₁₄ sphingosine standard under all four conditions(Table 1). However, the peak identified as C₁₄S-1-P demonstrated aslight difference from the C₁₄S-1-P standard under conditions whichexploit differences in charge (MeOH/10 mM KP/1 M TBAP, pH 7.2, 81:18:1).TABLE 1 Sphingolipid Identification C₁₄S C₁₄S-1-P Mobile Phase C₁₄S stdin extract C₁₄S-1-P std in extract MeOH/H₂O/1M 19.1 min 19.0 min 14.8min 14.8 min TBAP pH 4.8 82.1:17:0.9 MeOH/H₂O/1M 27.3 min 27.1 min 22.5min 22.1 min TBAP pH 4.8 79.1:20:0.9 MeOH/10 mM KP/1M 21.9 min 22.0 min18.3 min 17.2 min TBAP pH 5.5 81:18.1 MeOH/10 mM KP/1M 21.4 min 21.8 min15.0 min 17.1 min TBAP pH 7.2 81:18.1

[0140] This finding is likely to be due to a chemical modification ofthe phosphate group, since a phosphatase capable of dephosphorylatingthe C₁₄S-1-P standard does not recognize this substrate. Massspectroscopy is utilized to identify the phosphate group modification ofthis S-1-P species. Herein, this sphingolipid is referred to as“modified C₁₄S-1-P.”

Example 10 Characterization of Sphingolipid Species in the DrosophilaSPL Mutant

[0141] Differences in the quantity or type of sphingolipid speciespresent in mutant versus wild type adult flies and during various stagesof development was determined as described below.

[0142] Methods were as described in Example 9.

[0143] Results: The modified C₁₄S-1-P peak was ten-fold higher in theDrosophila SPL mutant than in the wild type (using an internal standardto normalize for extraction variation), supporting the notion that thephenotype of the SPL mutant may be due to abnormal accumulation ofphosphorylated sphingoid bases and resulting abnormalities in signaling.C₁₄ sphingosine was also increased in the mutant, but to a lesser extent(Table 2). No other peaks in the mutant demonstrated a significantdifference in comparison to wild type controls. TABLE 2 SphingolipidQuantification (nmol/200 mg flies) Line (n = 3) modified C₁₄S-1-P C₁₄SCanton S (wild type) 0.49 ± 0.07 2.61 ± 0.27 SPL mutant 4.49 ± 0.53 5.27± 0.73

Example 11 Characterization of the SPL Activity Encoded by ESTsLP04413/GH3783 and Which is Absent in Insertional Mutant 11393

[0144] Drosophila ESTs LP04413 and GH3783 encode a protein with stronghomology to other sphingosine phosphate lyases (SPL). Mutant 11393 whichdemonstrates the flight defect and dorsal longitudinal muscle (DLM)abnormalities described above in Example 7, contains a p-elementinsertion within this locus. Initial results using a standard SPL assayand a radiolabelled C₁₈DHS-1-P substrate indicated that Drosophila ESTsLP04413 and GH3783 encode an SPL, since expression restored SPL activityto a yeast SPL mutant. However, the activity conferred by the ESTexpression in yeast was not pronounced. Since Drosophila extractscontain C₁₄ sphingosine and a modified species of C₁₄S-1-P, it washypothesized that the C₁₈DHS-1-P was not a favorable substrate for themajor Drosophila lyase. Further, residual lyase activity observed in themutant indicated the presence of more than one SPL activity inDrosophila. Therefore, the optimal substrate of the SPL encoded by ESTsLP04413 and GH3783 was determined and this activity was differentiatedfrom other SPL activities in Drosophila.

[0145] Methods: Wild type (Canton S) and mutant whole fly extracts wereprepared by mechanical disruption. Standard SPL assays using C₁₈ DHS-1-Psubstrate were performed as previously described (Van Veldhoven, P. P.,and G. P. Mannaerts. 1991. Subcellular localization and membranetopology of sphingosine-1-phosphate lyase in rat liver. J Biol Chem.266:12502-12507). An HPLC-based SPL assay was established, to allow forvarious non-radioactive substrates to be evaluated. For this assay,C₁₄S-1-P, C₁₈DHS-1-P and modified C₁₄S-1-P were prepared by drying downthe lipid extract from 15 mg of 11939 flies, plus 200 pmol C₁₄S-1-Pstandard and 200 pmol C₁₈DHS-1-P standard. Lipids were resuspended in 25μl of 1% Triton X-100 in potassium phosphate buffer, pH 7.4. 175 μl ofreaction buffer (KP buffer, NaF, DTT, EDTA, sucrose) were added, andmixture was tip sonicated for 20 seconds, followed by addition of 50 μgof protein from whole cell extract of flies (CS or 11939) or Δdpl1:1cb4yeast overexpressing the fly lyase. Incubation proceeded for 1 hr at 37°C. Reaction was stopped by adding 175 μl of MeOH containing 0.2% aceticacid. The reaction was applied to STRATA C18 column in 40% MeOHcontaining 0.1% acetic acid. The column was washed with 600 μl of 40%MeOH containing 0.1% acetic acid. Lipids were eluted with 1 ml of 90%MeOH/10% 10 mM K-Phosphate, pH 7.2. Samples were dried and resuspendedin MeOH, treated with o-pthalaldehyde and injected on the HPLC. Thedegradation of S-1-P standards and modified C₁₄S-1-P were compared tostandards incubated in the absence of protein extracts.

[0146] Results: An activity which metabolizes modified C₁₄S-1-P ispresent in wild type fly extracts but is absent in the mutant flyextracts. Residual SPL activity does exist in the mutant fly. Thisactivity is distinct from that encoded by LP04413/GH3783, in that itmetabolizes C₁₄S-1-P and C₁₈DHS-1-P with an efficacy similar to orbetter than wild type. The pH curve of the residual SPL activity inmutant flies is identical to that seen in wild type flies (against aC₁₈DHS-1-P substrate), indicating that this activity is not disrupted inthe mutant.

Example 12 Further Characterization of the Drosophila melanogaster SPLMutant Phenotype

[0147] Adult SPL mutant flies demonstrated inability to fly and abnormalpatterning of indirect flight muscles. The adult SPL mutant fliesconsistently demonstrated abnormal patterning of DLMs, although thenumber of remaining DLMs varied in each mutant. In this Example, it wasdetermined whether the abnormal muscle development was limited to theadult fly, or whether the defect was also present at earlierdevelopmental stages.

[0148] Methods: Larval locomotor assay. Third instar larvae were placedon a clear agar substrate that overlays a grid. A light source at oneend provided a photactic stimulus. Distance traveled was scored duringthree minute trials. Larval muscle microscopy. Larvae were filletedduring the third instar and pinned with the dorsal cuticle down. Theviscera were removed to allow an unobstructed view of the body wallmuscles using polarized light. Muscles were refractile due to thepresence of filamentous arrays in each muscle fiber.

[0149] Results: 11393 mutant larvae demonstrated significant defects inlocomotion in comparison to wild type larvae, although phototacticresponse is intact. In all mutant larvae examined, the T2-dorsal obliquemuscles exhibited alterations in number and/or size. Fused,hypertrophied residual dorsal obliques were observed in the mutants.

[0150] Since the four pairs of dorsal obliques in thoracic segment twocreate scaffolds which give rise during pupation to the DLM structuresof the adult, it is likely that the developmental defect seen in theadult is the result of a process which begins much earlier, duringlarval development or embryogenesis.

Example 13 Human SPL Expression Patterns in Cancer

[0151] To determine if SPL expression is altered in human tumors, weutilized a cancer profiling array which contains more than 240 cDNApairs representing tumor tissue and corresponding normal tissue from thesame patient. By utilizing tissue pairs from one patient, differencesbetween gene expression in tumor and normal tissue which might be due toperson to person variability should not confound the interpretation ofresults. Additionally, each blot was normalized for loading using fourseparate housekeeping genes. Traditional hybridization techniques wereutilized to probe this cDNA blot with a 300 nucleotide 3′ fragment ofhuman SPL cDNA (SEQ ID NO:7), which was obtained from the previouslydescribed cloning experiments. Analysis of the array indicated that,whereas human SPL expression is matched closely in most tissue pairs, itis significantly reduced in colon cancer specimens, with a 50% reductionin expression in colloid cancer of the colon and 61% reduction inadenocarcinoma of the colon. Reduced SPL expression was also seen inadenocarcinaom of the uterus. None of the tumors in which SPL expressionis diminished demonstrate SK overexpression. Thus, altered SPLexpression is observed in primary human tumors. Therefore, modulatingthe activity of SPL protein either by altering gene expression orthrough direct action on the protein may provide a useful treatment forindividuals afflicted with an SPL-related cancer. Furthermore, SPLexpression serves as a useful diagnostic marker of cancer in humans.

[0152] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein for thepurpose of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention.

1 21 1 1770 DNA S. cerevisiae CDS (1)...(1770) 1 atg agt gga gta tca aataaa aca gta tca att aat ggt tgg tat ggc 48 Met Ser Gly Val Ser Asn LysThr Val Ser Ile Asn Gly Trp Tyr Gly 1 5 10 15 atg cca att cat tta ctaagg gaa gaa ggc gac ttt gcc cag ttt atg 96 Met Pro Ile His Leu Leu ArgGlu Glu Gly Asp Phe Ala Gln Phe Met 20 25 30 att cta acc atc aac gaa ttaaaa ata gcc ata cat ggt tac ctc aga 144 Ile Leu Thr Ile Asn Glu Leu LysIle Ala Ile His Gly Tyr Leu Arg 35 40 45 aat acc cca tgg tac aac atg ttgaag gat tat ttg ttt gtg atc ttt 192 Asn Thr Pro Trp Tyr Asn Met Leu LysAsp Tyr Leu Phe Val Ile Phe 50 55 60 tgt tac aag cta ata agt aat ttt ttttat ctg ttg aaa gtt tat ggg 240 Cys Tyr Lys Leu Ile Ser Asn Phe Phe TyrLeu Leu Lys Val Tyr Gly 65 70 75 80 ccg gtg agg tta gca gtg aga aca tacgag cat agt tcc aga aga ttg 288 Pro Val Arg Leu Ala Val Arg Thr Tyr GluHis Ser Ser Arg Arg Leu 85 90 95 ttt cgt tgg tta ttg gac tca cca ttt ttgagg ggt acc gta gaa aag 336 Phe Arg Trp Leu Leu Asp Ser Pro Phe Leu ArgGly Thr Val Glu Lys 100 105 110 gaa gtc aca aag gtc aaa caa tcg atc gaagac gaa cta att aga tcg 384 Glu Val Thr Lys Val Lys Gln Ser Ile Glu AspGlu Leu Ile Arg Ser 115 120 125 gac tct cag tta atg aat ttc cca cag ttgcca tcc aat ggg ata cct 432 Asp Ser Gln Leu Met Asn Phe Pro Gln Leu ProSer Asn Gly Ile Pro 130 135 140 cag gat gat gtt att gaa gag cta aat aaattg aac gac ttg ata cca 480 Gln Asp Asp Val Ile Glu Glu Leu Asn Lys LeuAsn Asp Leu Ile Pro 145 150 155 160 cat acc caa tgg aag gaa gga aag gtctct ggt gcc gtt tac cac ggt 528 His Thr Gln Trp Lys Glu Gly Lys Val SerGly Ala Val Tyr His Gly 165 170 175 ggt gat gat ttg atc cac tta caa acaatc gca tac gaa aaa tat tgc 576 Gly Asp Asp Leu Ile His Leu Gln Thr IleAla Tyr Glu Lys Tyr Cys 180 185 190 gtt gcc aat caa tta cat ccc gat gtcttt cct gcc gta cgt aaa atg 624 Val Ala Asn Gln Leu His Pro Asp Val PhePro Ala Val Arg Lys Met 195 200 205 gaa tcc gaa gtg gtt tct atg gtt ttaaga atg ttt aat gcc cct tct 672 Glu Ser Glu Val Val Ser Met Val Leu ArgMet Phe Asn Ala Pro Ser 210 215 220 gat aca ggt tgt ggt acc aca act tcaggt ggt aca gaa tcc ttg ctt 720 Asp Thr Gly Cys Gly Thr Thr Thr Ser GlyGly Thr Glu Ser Leu Leu 225 230 235 240 tta gca tgt ctg agc gct aaa atgtat gcc ctt cat cat cgt gga atc 768 Leu Ala Cys Leu Ser Ala Lys Met TyrAla Leu His His Arg Gly Ile 245 250 255 acc gaa cca gaa ata att gct cccgta act gca cat gct ggg ttt gac 816 Thr Glu Pro Glu Ile Ile Ala Pro ValThr Ala His Ala Gly Phe Asp 260 265 270 aaa gct gct tat tac ttt ggc atgaag cta cgc cac gtg gag cta gat 864 Lys Ala Ala Tyr Tyr Phe Gly Met LysLeu Arg His Val Glu Leu Asp 275 280 285 cca acg aca tat caa gtg gac ctggga aaa gtg aaa aaa ttc atc aat 912 Pro Thr Thr Tyr Gln Val Asp Leu GlyLys Val Lys Lys Phe Ile Asn 290 295 300 aag aac aca att tta ctg gtc ggttcc gct cca aac ttt cct cat ggt 960 Lys Asn Thr Ile Leu Leu Val Gly SerAla Pro Asn Phe Pro His Gly 305 310 315 320 att gcc gat gat att gaa ggattg ggt aaa ata gca caa aaa tat aaa 1008 Ile Ala Asp Asp Ile Glu Gly LeuGly Lys Ile Ala Gln Lys Tyr Lys 325 330 335 ctt cct tta cac gtc gac agttgt cta ggt tcc ttt att gtt tca ttt 1056 Leu Pro Leu His Val Asp Ser CysLeu Gly Ser Phe Ile Val Ser Phe 340 345 350 atg gaa aag gct ggt tac aaaaat ctg cca tta ctt gac ttt aga gtc 1104 Met Glu Lys Ala Gly Tyr Lys AsnLeu Pro Leu Leu Asp Phe Arg Val 355 360 365 ccg gga gtc acc tca ata tcatgt gac act cat aaa tat gga ttt gca 1152 Pro Gly Val Thr Ser Ile Ser CysAsp Thr His Lys Tyr Gly Phe Ala 370 375 380 cca aaa ggc tcg tca gtt ataatg tat aga aac agc gac tta cga atg 1200 Pro Lys Gly Ser Ser Val Ile MetTyr Arg Asn Ser Asp Leu Arg Met 385 390 395 400 cat cag tat tac gta aatcct gct tgg act ggc ggg tta tat ggc tct 1248 His Gln Tyr Tyr Val Asn ProAla Trp Thr Gly Gly Leu Tyr Gly Ser 405 410 415 cct aca tta gca ggg tccagg cct ggt gct att gtc gta ggt tgt tgg 1296 Pro Thr Leu Ala Gly Ser ArgPro Gly Ala Ile Val Val Gly Cys Trp 420 425 430 gcc act atg gtc aac atgggt gaa aat ggg tac att gag tcg tgc caa 1344 Ala Thr Met Val Asn Met GlyGlu Asn Gly Tyr Ile Glu Ser Cys Gln 435 440 445 gaa ata gtc ggt gca gcaatg aag ttt aaa aaa tac atc cag gaa aac 1392 Glu Ile Val Gly Ala Ala MetLys Phe Lys Lys Tyr Ile Gln Glu Asn 450 455 460 att cca gac ctg aat ataatg ggc aac cct aga tat tca gtc att tca 1440 Ile Pro Asp Leu Asn Ile MetGly Asn Pro Arg Tyr Ser Val Ile Ser 465 470 475 480 ttt tct tca aag accttg aac ata cac gaa cta tct gac agg ttg tcc 1488 Phe Ser Ser Lys Thr LeuAsn Ile His Glu Leu Ser Asp Arg Leu Ser 485 490 495 aag aaa ggc tgg catttc aat gcc cta caa aag ccg gtt gca cta cac 1536 Lys Lys Gly Trp His PheAsn Ala Leu Gln Lys Pro Val Ala Leu His 500 505 510 atg gcc ttc acg agattg agc gct cat gtt gtg gat gag atc tgc gac 1584 Met Ala Phe Thr Arg LeuSer Ala His Val Val Asp Glu Ile Cys Asp 515 520 525 att tta cgt act accgtg caa gag ttg aag agc gaa tca aat tct aaa 1632 Ile Leu Arg Thr Thr ValGln Glu Leu Lys Ser Glu Ser Asn Ser Lys 530 535 540 cca tcc cca gac ggaact agc gct cta tat ggt gtc gcc ggg agc gtt 1680 Pro Ser Pro Asp Gly ThrSer Ala Leu Tyr Gly Val Ala Gly Ser Val 545 550 555 560 aaa act gct ggcgtt gca gac aaa ttg att gtg gga ttc cta gac gca 1728 Lys Thr Ala Gly ValAla Asp Lys Leu Ile Val Gly Phe Leu Asp Ala 565 570 575 tta tac aag ttgggt cca gga gag gat acc gcc acc aag tag 1770 Leu Tyr Lys Leu Gly Pro GlyGlu Asp Thr Ala Thr Lys * 580 585 2 589 PRT S. cerevisiae 2 Met Ser GlyVal Ser Asn Lys Thr Val Ser Ile Asn Gly Trp Tyr Gly 1 5 10 15 Met ProIle His Leu Leu Arg Glu Glu Gly Asp Phe Ala Gln Phe Met 20 25 30 Ile LeuThr Ile Asn Glu Leu Lys Ile Ala Ile His Gly Tyr Leu Arg 35 40 45 Asn ThrPro Trp Tyr Asn Met Leu Lys Asp Tyr Leu Phe Val Ile Phe 50 55 60 Cys TyrLys Leu Ile Ser Asn Phe Phe Tyr Leu Leu Lys Val Tyr Gly 65 70 75 80 ProVal Arg Leu Ala Val Arg Thr Tyr Glu His Ser Ser Arg Arg Leu 85 90 95 PheArg Trp Leu Leu Asp Ser Pro Phe Leu Arg Gly Thr Val Glu Lys 100 105 110Glu Val Thr Lys Val Lys Gln Ser Ile Glu Asp Glu Leu Ile Arg Ser 115 120125 Asp Ser Gln Leu Met Asn Phe Pro Gln Leu Pro Ser Asn Gly Ile Pro 130135 140 Gln Asp Asp Val Ile Glu Glu Leu Asn Lys Leu Asn Asp Leu Ile Pro145 150 155 160 His Thr Gln Trp Lys Glu Gly Lys Val Ser Gly Ala Val TyrHis Gly 165 170 175 Gly Asp Asp Leu Ile His Leu Gln Thr Ile Ala Tyr GluLys Tyr Cys 180 185 190 Val Ala Asn Gln Leu His Pro Asp Val Phe Pro AlaVal Arg Lys Met 195 200 205 Glu Ser Glu Val Val Ser Met Val Leu Arg MetPhe Asn Ala Pro Ser 210 215 220 Asp Thr Gly Cys Gly Thr Thr Thr Ser GlyGly Thr Glu Ser Leu Leu 225 230 235 240 Leu Ala Cys Leu Ser Ala Lys MetTyr Ala Leu His His Arg Gly Ile 245 250 255 Thr Glu Pro Glu Ile Ile AlaPro Val Thr Ala His Ala Gly Phe Asp 260 265 270 Lys Ala Ala Tyr Tyr PheGly Met Lys Leu Arg His Val Glu Leu Asp 275 280 285 Pro Thr Thr Tyr GlnVal Asp Leu Gly Lys Val Lys Lys Phe Ile Asn 290 295 300 Lys Asn Thr IleLeu Leu Val Gly Ser Ala Pro Asn Phe Pro His Gly 305 310 315 320 Ile AlaAsp Asp Ile Glu Gly Leu Gly Lys Ile Ala Gln Lys Tyr Lys 325 330 335 LeuPro Leu His Val Asp Ser Cys Leu Gly Ser Phe Ile Val Ser Phe 340 345 350Met Glu Lys Ala Gly Tyr Lys Asn Leu Pro Leu Leu Asp Phe Arg Val 355 360365 Pro Gly Val Thr Ser Ile Ser Cys Asp Thr His Lys Tyr Gly Phe Ala 370375 380 Pro Lys Gly Ser Ser Val Ile Met Tyr Arg Asn Ser Asp Leu Arg Met385 390 395 400 His Gln Tyr Tyr Val Asn Pro Ala Trp Thr Gly Gly Leu TyrGly Ser 405 410 415 Pro Thr Leu Ala Gly Ser Arg Pro Gly Ala Ile Val ValGly Cys Trp 420 425 430 Ala Thr Met Val Asn Met Gly Glu Asn Gly Tyr IleGlu Ser Cys Gln 435 440 445 Glu Ile Val Gly Ala Ala Met Lys Phe Lys LysTyr Ile Gln Glu Asn 450 455 460 Ile Pro Asp Leu Asn Ile Met Gly Asn ProArg Tyr Ser Val Ile Ser 465 470 475 480 Phe Ser Ser Lys Thr Leu Asn IleHis Glu Leu Ser Asp Arg Leu Ser 485 490 495 Lys Lys Gly Trp His Phe AsnAla Leu Gln Lys Pro Val Ala Leu His 500 505 510 Met Ala Phe Thr Arg LeuSer Ala His Val Val Asp Glu Ile Cys Asp 515 520 525 Ile Leu Arg Thr ThrVal Gln Glu Leu Lys Ser Glu Ser Asn Ser Lys 530 535 540 Pro Ser Pro AspGly Thr Ser Ala Leu Tyr Gly Val Ala Gly Ser Val 545 550 555 560 Lys ThrAla Gly Val Ala Asp Lys Leu Ile Val Gly Phe Leu Asp Ala 565 570 575 LeuTyr Lys Leu Gly Pro Gly Glu Asp Thr Ala Thr Lys 580 585 3 1629 DNA C.elegans CDS (1)...(1629) 3 atg gat ttt gca ctg gag caa tat cat agt gcaaag gat ttg tta ata 48 Met Asp Phe Ala Leu Glu Gln Tyr His Ser Ala LysAsp Leu Leu Ile 1 5 10 15 ttt gag ctt cga aag ttc aat cca att gtt ctggtt tct agt act att 96 Phe Glu Leu Arg Lys Phe Asn Pro Ile Val Leu ValSer Ser Thr Ile 20 25 30 gtt gca aca tac gta ctc acc aat ctg aga cat atgcat tta gat gaa 144 Val Ala Thr Tyr Val Leu Thr Asn Leu Arg His Met HisLeu Asp Glu 35 40 45 atg ggc atc cgg aaa cgt ttg agc act tgg ttt ttc accact gta aag 192 Met Gly Ile Arg Lys Arg Leu Ser Thr Trp Phe Phe Thr ThrVal Lys 50 55 60 cgt gtg cct ttc atc agg aaa atg att gac aaa caa cta aacgaa gta 240 Arg Val Pro Phe Ile Arg Lys Met Ile Asp Lys Gln Leu Asn GluVal 65 70 75 80 aag gac gag ctt gag aaa agt ctg aga att gtg gat cga agcacc gaa 288 Lys Asp Glu Leu Glu Lys Ser Leu Arg Ile Val Asp Arg Ser ThrGlu 85 90 95 tac ttc act aca atc cca agc cat tca gtt gga aga act gaa gtactt 336 Tyr Phe Thr Thr Ile Pro Ser His Ser Val Gly Arg Thr Glu Val Leu100 105 110 cgc ctt gct gcc atc tat gat gat ttg gaa gga cca gct ttt ttggaa 384 Arg Leu Ala Ala Ile Tyr Asp Asp Leu Glu Gly Pro Ala Phe Leu Glu115 120 125 gga aga gta tct gga gca gtc ttc aat aga gaa gac gac aag gacgaa 432 Gly Arg Val Ser Gly Ala Val Phe Asn Arg Glu Asp Asp Lys Asp Glu130 135 140 cgg gag atg tat gag gag gtg ttc gga aaa ttt gcc tgg acc aaccca 480 Arg Glu Met Tyr Glu Glu Val Phe Gly Lys Phe Ala Trp Thr Asn Pro145 150 155 160 ctt tgg cca aaa ttg ttc cct gga gtg aga atc atg gag gctgaa gtt 528 Leu Trp Pro Lys Leu Phe Pro Gly Val Arg Ile Met Glu Ala GluVal 165 170 175 gtt cgc atg tgt tgt aat atg atg aat gga gat tcg gag acatgt gga 576 Val Arg Met Cys Cys Asn Met Met Asn Gly Asp Ser Glu Thr CysGly 180 185 190 act atg tca act ggt gga tcc att tca att ctt ttg gcg tgcctg gct 624 Thr Met Ser Thr Gly Gly Ser Ile Ser Ile Leu Leu Ala Cys LeuAla 195 200 205 cat cgt aat cgt ctt ttg aaa aga gga gaa aag tac aca gagatg att 672 His Arg Asn Arg Leu Leu Lys Arg Gly Glu Lys Tyr Thr Glu MetIle 210 215 220 gtc cca tca tcc gtc cat gca gcg ttc ttc aaa gct gcc gaatgt ttc 720 Val Pro Ser Ser Val His Ala Ala Phe Phe Lys Ala Ala Glu CysPhe 225 230 235 240 cgt atc aaa gtt cgc aag att cca gtt gat cct gtt actttc aaa gta 768 Arg Ile Lys Val Arg Lys Ile Pro Val Asp Pro Val Thr PheLys Val 245 250 255 gac ctt gtc aaa atg aaa gcc gca att aac aag aga acatgt atg tta 816 Asp Leu Val Lys Met Lys Ala Ala Ile Asn Lys Arg Thr CysMet Leu 260 265 270 gtt gga tct gct cca aac ttt cca ttt gga act gtt gatgac att gaa 864 Val Gly Ser Ala Pro Asn Phe Pro Phe Gly Thr Val Asp AspIle Glu 275 280 285 gct att gga cag cta gga ctt gaa tat gac atc cca gttcat gtt gat 912 Ala Ile Gly Gln Leu Gly Leu Glu Tyr Asp Ile Pro Val HisVal Asp 290 295 300 gct tgt ctt ggt ggt ttc ctt ctt cca ttc ctt gaa gaagac gag att 960 Ala Cys Leu Gly Gly Phe Leu Leu Pro Phe Leu Glu Glu AspGlu Ile 305 310 315 320 cgc tat gac ttc cgt gtt cct ggt gta tct tcg atttct gca gat agt 1008 Arg Tyr Asp Phe Arg Val Pro Gly Val Ser Ser Ile SerAla Asp Ser 325 330 335 cac aaa tac gga ctc gct cca aag ggg tca tca gttgtt ctt tat cgc 1056 His Lys Tyr Gly Leu Ala Pro Lys Gly Ser Ser Val ValLeu Tyr Arg 340 345 350 aat aag gaa ctt ctt cat aat cag tac ttc tgt gatgct gat tgg caa 1104 Asn Lys Glu Leu Leu His Asn Gln Tyr Phe Cys Asp AlaAsp Trp Gln 355 360 365 gga ggt atc tat gca tcg gct act atg gaa gga tcacgc gct ggg cac 1152 Gly Gly Ile Tyr Ala Ser Ala Thr Met Glu Gly Ser ArgAla Gly His 370 375 380 aac att gca ctt tgc tgg gcc gca atg ctt tat cacgct cag gaa gga 1200 Asn Ile Ala Leu Cys Trp Ala Ala Met Leu Tyr His AlaGln Glu Gly 385 390 395 400 tac aag gcc aat gct aga aag att gtt gac actaca aga aag att aga 1248 Tyr Lys Ala Asn Ala Arg Lys Ile Val Asp Thr ThrArg Lys Ile Arg 405 410 415 aat gga ctt tca aac att aag gga atc aaa ttacaa ggg cca agt gat 1296 Asn Gly Leu Ser Asn Ile Lys Gly Ile Lys Leu GlnGly Pro Ser Asp 420 425 430 gtt tgt att gtt agc tgg aca acc aat gat ggagtt gaa ctc tac aga 1344 Val Cys Ile Val Ser Trp Thr Thr Asn Asp Gly ValGlu Leu Tyr Arg 435 440 445 ttc cat aac ttc atg aag gaa aaa cat tgg caactg aat gga ctt caa 1392 Phe His Asn Phe Met Lys Glu Lys His Trp Gln LeuAsn Gly Leu Gln 450 455 460 ttc cca gct gga gtt cat atc atg gtc act atgaat cat act cat cct 1440 Phe Pro Ala Gly Val His Ile Met Val Thr Met AsnHis Thr His Pro 465 470 475 480 gga ctc gct gaa gct ttc gtc gcc gat tgcaga gct gca gtt gag ttt 1488 Gly Leu Ala Glu Ala Phe Val Ala Asp Cys ArgAla Ala Val Glu Phe 485 490 495 gtc aaa agc cac aaa cca tcg gaa tcc gacaag aca agt gaa gca gcc 1536 Val Lys Ser His Lys Pro Ser Glu Ser Asp LysThr Ser Glu Ala Ala 500 505 510 atc tac gga ctt gct caa agt att cca gaccga tcg ctt gtt cac gag 1584 Ile Tyr Gly Leu Ala Gln Ser Ile Pro Asp ArgSer Leu Val His Glu 515 520 525 ttt gct cac agc tat atc gat gct gtt tatgct tta aca gag tga 1629 Phe Ala His Ser Tyr Ile Asp Ala Val Tyr Ala LeuThr Glu * 530 535 540 4 542 PRT C. elegans 4 Met Asp Phe Ala Leu Glu GlnTyr His Ser Ala Lys Asp Leu Leu Ile 1 5 10 15 Phe Glu Leu Arg Lys PheAsn Pro Ile Val Leu Val Ser Ser Thr Ile 20 25 30 Val Ala Thr Tyr Val LeuThr Asn Leu Arg His Met His Leu Asp Glu 35 40 45 Met Gly Ile Arg Lys ArgLeu Ser Thr Trp Phe Phe Thr Thr Val Lys 50 55 60 Arg Val Pro Phe Ile ArgLys Met Ile Asp Lys Gln Leu Asn Glu Val 65 70 75 80 Lys Asp Glu Leu GluLys Ser Leu Arg Ile Val Asp Arg Ser Thr Glu 85 90 95 Tyr Phe Thr Thr IlePro Ser His Ser Val Gly Arg Thr Glu Val Leu 100 105 110 Arg Leu Ala AlaIle Tyr Asp Asp Leu Glu Gly Pro Ala Phe Leu Glu 115 120 125 Gly Arg ValSer Gly Ala Val Phe Asn Arg Glu Asp Asp Lys Asp Glu 130 135 140 Arg GluMet Tyr Glu Glu Val Phe Gly Lys Phe Ala Trp Thr Asn Pro 145 150 155 160Leu Trp Pro Lys Leu Phe Pro Gly Val Arg Ile Met Glu Ala Glu Val 165 170175 Val Arg Met Cys Cys Asn Met Met Asn Gly Asp Ser Glu Thr Cys Gly 180185 190 Thr Met Ser Thr Gly Gly Ser Ile Ser Ile Leu Leu Ala Cys Leu Ala195 200 205 His Arg Asn Arg Leu Leu Lys Arg Gly Glu Lys Tyr Thr Glu MetIle 210 215 220 Val Pro Ser Ser Val His Ala Ala Phe Phe Lys Ala Ala GluCys Phe 225 230 235 240 Arg Ile Lys Val Arg Lys Ile Pro Val Asp Pro ValThr Phe Lys Val 245 250 255 Asp Leu Val Lys Met Lys Ala Ala Ile Asn LysArg Thr Cys Met Leu 260 265 270 Val Gly Ser Ala Pro Asn Phe Pro Phe GlyThr Val Asp Asp Ile Glu 275 280 285 Ala Ile Gly Gln Leu Gly Leu Glu TyrAsp Ile Pro Val His Val Asp 290 295 300 Ala Cys Leu Gly Gly Phe Leu LeuPro Phe Leu Glu Glu Asp Glu Ile 305 310 315 320 Arg Tyr Asp Phe Arg ValPro Gly Val Ser Ser Ile Ser Ala Asp Ser 325 330 335 His Lys Tyr Gly LeuAla Pro Lys Gly Ser Ser Val Val Leu Tyr Arg 340 345 350 Asn Lys Glu LeuLeu His Asn Gln Tyr Phe Cys Asp Ala Asp Trp Gln 355 360 365 Gly Gly IleTyr Ala Ser Ala Thr Met Glu Gly Ser Arg Ala Gly His 370 375 380 Asn IleAla Leu Cys Trp Ala Ala Met Leu Tyr His Ala Gln Glu Gly 385 390 395 400Tyr Lys Ala Asn Ala Arg Lys Ile Val Asp Thr Thr Arg Lys Ile Arg 405 410415 Asn Gly Leu Ser Asn Ile Lys Gly Ile Lys Leu Gln Gly Pro Ser Asp 420425 430 Val Cys Ile Val Ser Trp Thr Thr Asn Asp Gly Val Glu Leu Tyr Arg435 440 445 Phe His Asn Phe Met Lys Glu Lys His Trp Gln Leu Asn Gly LeuGln 450 455 460 Phe Pro Ala Gly Val His Ile Met Val Thr Met Asn His ThrHis Pro 465 470 475 480 Gly Leu Ala Glu Ala Phe Val Ala Asp Cys Arg AlaAla Val Glu Phe 485 490 495 Val Lys Ser His Lys Pro Ser Glu Ser Asp LysThr Ser Glu Ala Ala 500 505 510 Ile Tyr Gly Leu Ala Gln Ser Ile Pro AspArg Ser Leu Val His Glu 515 520 525 Phe Ala His Ser Tyr Ile Asp Ala ValTyr Ala Leu Thr Glu 530 535 540 5 1707 DNA Mus musculus CDS (1)...(1707)5 atg ccc gga acc gac ctc ctc aag ctg aag gac ttc gag cct tat ttg 48 MetPro Gly Thr Asp Leu Leu Lys Leu Lys Asp Phe Glu Pro Tyr Leu 1 5 10 15gag att ttg gaa tct tat tcc aca aaa gcc aag aat tat gtg aat gga 96 GluIle Leu Glu Ser Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 tattgc acc aaa tat gag ccc tgg cag ctc att gcg tgg agt gtc ctg 144 Tyr CysThr Lys Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val Leu 35 40 45 tgt actctg ctg ata gtc tgg gtg tat gag ctt atc ttc cag cca gag 192 Cys Thr LeuLeu Ile Val Trp Val Tyr Glu Leu Ile Phe Gln Pro Glu 50 55 60 agt tta tggtct cgg ttt aaa aaa aaa tta ttt aag ctt atc agg aag 240 Ser Leu Trp SerArg Phe Lys Lys Lys Leu Phe Lys Leu Ile Arg Lys 65 70 75 80 atg cca tttatt gga cgt aag atc gaa caa cag gtg agc aaa gcc aag 288 Met Pro Phe IleGly Arg Lys Ile Glu Gln Gln Val Ser Lys Ala Lys 85 90 95 aag gat ctt gtcaag aac atg cca ttc cta aag gtg gac aag gat tat 336 Lys Asp Leu Val LysAsn Met Pro Phe Leu Lys Val Asp Lys Asp Tyr 100 105 110 gtg aaa act ctgcct gct cag ggt atg ggc aca gct gag gtt ctg gag 384 Val Lys Thr Leu ProAla Gln Gly Met Gly Thr Ala Glu Val Leu Glu 115 120 125 aga ctc aag gagtac agc tcc atg gat ggt tcc tgg caa gaa ggg aaa 432 Arg Leu Lys Glu TyrSer Ser Met Asp Gly Ser Trp Gln Glu Gly Lys 130 135 140 gcc tca gga gctgtg tac aat ggg gaa ccg aag ctc acg gag ctg ctg 480 Ala Ser Gly Ala ValTyr Asn Gly Glu Pro Lys Leu Thr Glu Leu Leu 145 150 155 160 gtg cag gcttat gga gaa ttc acg tgg agc aat cca ctg cat cca gat 528 Val Gln Ala TyrGly Glu Phe Thr Trp Ser Asn Pro Leu His Pro Asp 165 170 175 atc ttc cctgga ttg cgg aag tta gag gca gaa atc gtt agg atg act 576 Ile Phe Pro GlyLeu Arg Lys Leu Glu Ala Glu Ile Val Arg Met Thr 180 185 190 tgt tcc ctcttc aat ggg gga cca gat tcc tgt gga tgt gtg act tct 624 Cys Ser Leu PheAsn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr Ser 195 200 205 ggg gga acggaa agc atc ctg atg gcc tgc aaa gct tac cgg gac ttg 672 Gly Gly Thr GluSer Ile Leu Met Ala Cys Lys Ala Tyr Arg Asp Leu 210 215 220 gcg tta gagaag ggg atc aaa act cca gaa att gtg gct ccc gag agt 720 Ala Leu Glu LysGly Ile Lys Thr Pro Glu Ile Val Ala Pro Glu Ser 225 230 235 240 gcc catgct gca ttc gac aaa gca gct cat tat ttt ggg atg aag att 768 Ala His AlaAla Phe Asp Lys Ala Ala His Tyr Phe Gly Met Lys Ile 245 250 255 gtc cgagtt gca ctg aaa aag aac atg gag gtg gat gtg cag gca atg 816 Val Arg ValAla Leu Lys Lys Asn Met Glu Val Asp Val Gln Ala Met 260 265 270 aag agagcc atc tcc agg aac aca gct atg ctg gtc tgt tct acc cca 864 Lys Arg AlaIle Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275 280 285 cag tttcct cat ggt gtg atg gat cct gtc ccc gaa gtg gcc aag tta 912 Gln Phe ProHis Gly Val Met Asp Pro Val Pro Glu Val Ala Lys Leu 290 295 300 act gtcaga tat aaa atc cca ctc cat gtg gat gct tgt ctg ggg ggc 960 Thr Val ArgTyr Lys Ile Pro Leu His Val Asp Ala Cys Leu Gly Gly 305 310 315 320 ttcctc att gtc ttc atg gag aaa gca ggg tac cca ctg gag aaa cca 1008 Phe LeuIle Val Phe Met Glu Lys Ala Gly Tyr Pro Leu Glu Lys Pro 325 330 335 tttgat ttc cgg gtg aaa ggt gtg acc agc att tca gca gat act cat 1056 Phe AspPhe Arg Val Lys Gly Val Thr Ser Ile Ser Ala Asp Thr His 340 345 350 aagtat ggc tat gct cct aaa ggt tca tca gtg gtg atg tac tct aac 1104 Lys TyrGly Tyr Ala Pro Lys Gly Ser Ser Val Val Met Tyr Ser Asn 355 360 365 gagaag tac agg acg tac cag ttc ttt gtt ggt gca gac tgg caa ggt 1152 Glu LysTyr Arg Thr Tyr Gln Phe Phe Val Gly Ala Asp Trp Gln Gly 370 375 380 ggtgtc tac gca tct cca agc ata gct ggc tca cgg cct ggt ggc atc 1200 Gly ValTyr Ala Ser Pro Ser Ile Ala Gly Ser Arg Pro Gly Gly Ile 385 390 395 400att gca gcc tgt tgg gcg gcc ttg atg cac ttc ggt gag aac ggc tat 1248 IleAla Ala Cys Trp Ala Ala Leu Met His Phe Gly Glu Asn Gly Tyr 405 410 415gtt gaa gct acc aaa cag atc atc aaa act gct cgc ttc ctg aag tca 1296 ValGlu Ala Thr Lys Gln Ile Ile Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430gaa ctg gaa aac atc aaa aac atc ttc att ttc ggt gat cct caa ttg 1344 GluLeu Glu Asn Ile Lys Asn Ile Phe Ile Phe Gly Asp Pro Gln Leu 435 440 445tca gtt att gct ctg gga tcc aac gat ttt gac att tac cga cta tct 1392 SerVal Ile Ala Leu Gly Ser Asn Asp Phe Asp Ile Tyr Arg Leu Ser 450 455 460aat atg atg tct gct aag ggg tgg aat ttt aac tac ctg cag ttc cca 1440 AsnMet Met Ser Ala Lys Gly Trp Asn Phe Asn Tyr Leu Gln Phe Pro 465 470 475480 aga agc att cat ttc tgc att acg tta gta cat act cgg aag cga gtg 1488Arg Ser Ile His Phe Cys Ile Thr Leu Val His Thr Arg Lys Arg Val 485 490495 gcg atc cag ttc cta aag gat atc cgg gaa tca gtc aca caa atc atg 1536Ala Ile Gln Phe Leu Lys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 500 505510 aag aat cct aaa gct aag acc aca gga atg ggt gcc atc tat ggc atg 1584Lys Asn Pro Lys Ala Lys Thr Thr Gly Met Gly Ala Ile Tyr Gly Met 515 520525 gcc cag gca acc att gac agg aag ctg gtt gca gaa ata tcc tcc gtc 1632Ala Gln Ala Thr Ile Asp Arg Lys Leu Val Ala Glu Ile Ser Ser Val 530 535540 ttc ttg gac tgc ctt tat act acg gac ccc gtg act cag ggc aac cag 1680Phe Leu Asp Cys Leu Tyr Thr Thr Asp Pro Val Thr Gln Gly Asn Gln 545 550555 560 atg aac ggt tct cca aag ccc cgc tga 1707 Met Asn Gly Ser Pro LysPro Arg * 565 6 568 PRT Mus musculus 6 Met Pro Gly Thr Asp Leu Leu LysLeu Lys Asp Phe Glu Pro Tyr Leu 1 5 10 15 Glu Ile Leu Glu Ser Tyr SerThr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 Tyr Cys Thr Lys Tyr Glu ProTrp Gln Leu Ile Ala Trp Ser Val Leu 35 40 45 Cys Thr Leu Leu Ile Val TrpVal Tyr Glu Leu Ile Phe Gln Pro Glu 50 55 60 Ser Leu Trp Ser Arg Phe LysLys Lys Leu Phe Lys Leu Ile Arg Lys 65 70 75 80 Met Pro Phe Ile Gly ArgLys Ile Glu Gln Gln Val Ser Lys Ala Lys 85 90 95 Lys Asp Leu Val Lys AsnMet Pro Phe Leu Lys Val Asp Lys Asp Tyr 100 105 110 Val Lys Thr Leu ProAla Gln Gly Met Gly Thr Ala Glu Val Leu Glu 115 120 125 Arg Leu Lys GluTyr Ser Ser Met Asp Gly Ser Trp Gln Glu Gly Lys 130 135 140 Ala Ser GlyAla Val Tyr Asn Gly Glu Pro Lys Leu Thr Glu Leu Leu 145 150 155 160 ValGln Ala Tyr Gly Glu Phe Thr Trp Ser Asn Pro Leu His Pro Asp 165 170 175Ile Phe Pro Gly Leu Arg Lys Leu Glu Ala Glu Ile Val Arg Met Thr 180 185190 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr Ser 195200 205 Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys Ala Tyr Arg Asp Leu210 215 220 Ala Leu Glu Lys Gly Ile Lys Thr Pro Glu Ile Val Ala Pro GluSer 225 230 235 240 Ala His Ala Ala Phe Asp Lys Ala Ala His Tyr Phe GlyMet Lys Ile 245 250 255 Val Arg Val Ala Leu Lys Lys Asn Met Glu Val AspVal Gln Ala Met 260 265 270 Lys Arg Ala Ile Ser Arg Asn Thr Ala Met LeuVal Cys Ser Thr Pro 275 280 285 Gln Phe Pro His Gly Val Met Asp Pro ValPro Glu Val Ala Lys Leu 290 295 300 Thr Val Arg Tyr Lys Ile Pro Leu HisVal Asp Ala Cys Leu Gly Gly 305 310 315 320 Phe Leu Ile Val Phe Met GluLys Ala Gly Tyr Pro Leu Glu Lys Pro 325 330 335 Phe Asp Phe Arg Val LysGly Val Thr Ser Ile Ser Ala Asp Thr His 340 345 350 Lys Tyr Gly Tyr AlaPro Lys Gly Ser Ser Val Val Met Tyr Ser Asn 355 360 365 Glu Lys Tyr ArgThr Tyr Gln Phe Phe Val Gly Ala Asp Trp Gln Gly 370 375 380 Gly Val TyrAla Ser Pro Ser Ile Ala Gly Ser Arg Pro Gly Gly Ile 385 390 395 400 IleAla Ala Cys Trp Ala Ala Leu Met His Phe Gly Glu Asn Gly Tyr 405 410 415Val Glu Ala Thr Lys Gln Ile Ile Lys Thr Ala Arg Phe Leu Lys Ser 420 425430 Glu Leu Glu Asn Ile Lys Asn Ile Phe Ile Phe Gly Asp Pro Gln Leu 435440 445 Ser Val Ile Ala Leu Gly Ser Asn Asp Phe Asp Ile Tyr Arg Leu Ser450 455 460 Asn Met Met Ser Ala Lys Gly Trp Asn Phe Asn Tyr Leu Gln PhePro 465 470 475 480 Arg Ser Ile His Phe Cys Ile Thr Leu Val His Thr ArgLys Arg Val 485 490 495 Ala Ile Gln Phe Leu Lys Asp Ile Arg Glu Ser ValThr Gln Ile Met 500 505 510 Lys Asn Pro Lys Ala Lys Thr Thr Gly Met GlyAla Ile Tyr Gly Met 515 520 525 Ala Gln Ala Thr Ile Asp Arg Lys Leu ValAla Glu Ile Ser Ser Val 530 535 540 Phe Leu Asp Cys Leu Tyr Thr Thr AspPro Val Thr Gln Gly Asn Gln 545 550 555 560 Met Asn Gly Ser Pro Lys ProArg 565 7 1707 DNA Homo sapiens CDS (1)...(1707) 7 atg cct agc aca gacctt ctg atg ttg aag gcc ttt gag ccc tac tta 48 Met Pro Ser Thr Asp LeuLeu Met Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 10 15 gag att ttg gaa gtatac tcc aca aaa gcc aag aat tat gta aat gga 96 Glu Ile Leu Glu Val TyrSer Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 cat tgc acc aag tat gagccc tgg cag cta att gca tgg agt gtc gtg 144 His Cys Thr Lys Tyr Glu ProTrp Gln Leu Ile Ala Trp Ser Val Val 35 40 45 tgg acc ctg ctg ata gtc tgggga tat gag ttt gtc ttc cag cca gag 192 Trp Thr Leu Leu Ile Val Trp GlyTyr Glu Phe Val Phe Gln Pro Glu 50 55 60 agt tta tgg tca agg ttt aaa aagaaa tgt ttt aag ctc acc agg aag 240 Ser Leu Trp Ser Arg Phe Lys Lys LysCys Phe Lys Leu Thr Arg Lys 65 70 75 80 atg ccc att att ggt cgt aag attcaa gac aag ttg aac aag acc aag 288 Met Pro Ile Ile Gly Arg Lys Ile GlnAsp Lys Leu Asn Lys Thr Lys 85 90 95 gat gat att agc aag aac atg tca ttcctg aaa gtg gac aaa gag tat 336 Asp Asp Ile Ser Lys Asn Met Ser Phe LeuLys Val Asp Lys Glu Tyr 100 105 110 gtg aaa gct tta ccc tcc cag ggt ctgagc tca tct gct gtt ttg gag 384 Val Lys Ala Leu Pro Ser Gln Gly Leu SerSer Ser Ala Val Leu Glu 115 120 125 aaa ctt aag gag tac agc tct atg gacgcc ttc tgg caa gag ggg aga 432 Lys Leu Lys Glu Tyr Ser Ser Met Asp AlaPhe Trp Gln Glu Gly Arg 130 135 140 gcc tct gga aca gtg tac agt ggg gaggag aag ctc act gag ctc ctt 480 Ala Ser Gly Thr Val Tyr Ser Gly Glu GluLys Leu Thr Glu Leu Leu 145 150 155 160 gtg aag gct tat gga gat ttt gcatgg agt aac ccc ctg cat cca gat 528 Val Lys Ala Tyr Gly Asp Phe Ala TrpSer Asn Pro Leu His Pro Asp 165 170 175 atc ttc cca gga cta cgc aag atagag gca gaa att gtg agg ata gct 576 Ile Phe Pro Gly Leu Arg Lys Ile GluAla Glu Ile Val Arg Ile Ala 180 185 190 tgt tcc ctg ttc aat ggg gga ccagat tcg tgt gga tgt gtg act tct 624 Cys Ser Leu Phe Asn Gly Gly Pro AspSer Cys Gly Cys Val Thr Ser 195 200 205 ggg gga aca gaa agc ata ctc atggcc tgc aaa gca tgt cgg gat ctg 672 Gly Gly Thr Glu Ser Ile Leu Met AlaCys Lys Ala Cys Arg Asp Leu 210 215 220 gcc ttt gag aag ggg atc aaa actcca gaa att gtg gct ccc caa agt 720 Ala Phe Glu Lys Gly Ile Lys Thr ProGlu Ile Val Ala Pro Gln Ser 225 230 235 240 gcc cat gct gca ttt aac aaagca gcc agt tac ttt ggg atg aag att 768 Ala His Ala Ala Phe Asn Lys AlaAla Ser Tyr Phe Gly Met Lys Ile 245 250 255 gtg cgg gtc cca ttg acg aagatg atg gag gtg gat gtg agg gca atg 816 Val Arg Val Pro Leu Thr Lys MetMet Glu Val Asp Val Arg Ala Met 260 265 270 aga aga gct atc tcc agg aacact gcc atg ctc gtc tgt tct acc cca 864 Arg Arg Ala Ile Ser Arg Asn ThrAla Met Leu Val Cys Ser Thr Pro 275 280 285 cag ttt cct cat ggt gta atagat cct gtc cct gaa gtg gcc aag ctg 912 Gln Phe Pro His Gly Val Ile AspPro Val Pro Glu Val Ala Lys Leu 290 295 300 gct gtc aaa tac aaa ata cccctt cat gtc gac gct tgt ctg gga ggc 960 Ala Val Lys Tyr Lys Ile Pro LeuHis Val Asp Ala Cys Leu Gly Gly 305 310 315 320 ttc ctc atc gtc ttt atggag aaa gca gga tac cca ctg gag cac cca 1008 Phe Leu Ile Val Phe Met GluLys Ala Gly Tyr Pro Leu Glu His Pro 325 330 335 ttt gat ttc cgg gtg aaaggt gta acc agc att tca gct gac acc cat 1056 Phe Asp Phe Arg Val Lys GlyVal Thr Ser Ile Ser Ala Asp Thr His 340 345 350 aag tat ggc tat gcc ccaaaa ggc tca tca ttg gtg ttg tat agt gac 1104 Lys Tyr Gly Tyr Ala Pro LysGly Ser Ser Leu Val Leu Tyr Ser Asp 355 360 365 aag aag tac agg aac tatcag ttc ttc gtc gat aca gat tgg cag ggt 1152 Lys Lys Tyr Arg Asn Tyr GlnPhe Phe Val Asp Thr Asp Trp Gln Gly 370 375 380 ggc atc tat gct tcc ccaacc atc gca ggc tca cgg cct ggt ggc att 1200 Gly Ile Tyr Ala Ser Pro ThrIle Ala Gly Ser Arg Pro Gly Gly Ile 385 390 395 400 agc gca gcc tgt tgggct gcc ttg atg cac ttc ggt gag aac ggc tat 1248 Ser Ala Ala Cys Trp AlaAla Leu Met His Phe Gly Glu Asn Gly Tyr 405 410 415 gtt gaa gct acc aaacag atc atc aaa act gct cgc ttc ctc aag tca 1296 Val Glu Ala Thr Lys GlnIle Ile Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430 gaa ctg gaa aat atcaaa ggc atc ttt gtt ttt ggg aat ccc caa ttg 1344 Glu Leu Glu Asn Ile LysGly Ile Phe Val Phe Gly Asn Pro Gln Leu 435 440 445 tca ctc att gct ctggga tcc cgt gat ttt gac atc tac cga cta tca 1392 Ser Leu Ile Ala Leu GlySer Arg Asp Phe Asp Ile Tyr Arg Leu Ser 450 455 460 aac ctg atg act gctaag ggg tgg aac ttg aac cag ttg cag ttc cca 1440 Asn Leu Met Thr Ala LysGly Trp Asn Leu Asn Gln Leu Gln Phe Pro 465 470 475 480 ccc agt att catttc tgc atc aca tta cta cac gcc cgg aaa cga gta 1488 Pro Ser Ile His PheCys Ile Thr Leu Leu His Ala Arg Lys Arg Val 485 490 495 gct ata caa ttccta aag gac att cga gaa tct gtc act caa atc atg 1536 Ala Ile Gln Phe LeuLys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 500 505 510 aag aat cct aaagcg aag acc aca gga atg ggt gcc atc tat gcc atg 1584 Lys Asn Pro Lys AlaLys Thr Thr Gly Met Gly Ala Ile Tyr Ala Met 515 520 525 gcc cag aca actgtt gac agg aat atg gtt gca gaa ttg tcc tca gtc 1632 Ala Gln Thr Thr ValAsp Arg Asn Met Val Ala Glu Leu Ser Ser Val 530 535 540 ttc ttg gac agcttg tac agc acc gac act gtc acc cag ggc agc cag 1680 Phe Leu Asp Ser LeuTyr Ser Thr Asp Thr Val Thr Gln Gly Ser Gln 545 550 555 560 atg aat ggttct cca aaa ccc cac tga 1707 Met Asn Gly Ser Pro Lys Pro His * 565 8 568PRT Homo sapiens 8 Met Pro Ser Thr Asp Leu Leu Met Leu Lys Ala Phe GluPro Tyr Leu 1 5 10 15 Glu Ile Leu Glu Val Tyr Ser Thr Lys Ala Lys AsnTyr Val Asn Gly 20 25 30 His Cys Thr Lys Tyr Glu Pro Trp Gln Leu Ile AlaTrp Ser Val Val 35 40 45 Trp Thr Leu Leu Ile Val Trp Gly Tyr Glu Phe ValPhe Gln Pro Glu 50 55 60 Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys Phe LysLeu Thr Arg Lys 65 70 75 80 Met Pro Ile Ile Gly Arg Lys Ile Gln Asp LysLeu Asn Lys Thr Lys 85 90 95 Asp Asp Ile Ser Lys Asn Met Ser Phe Leu LysVal Asp Lys Glu Tyr 100 105 110 Val Lys Ala Leu Pro Ser Gln Gly Leu SerSer Ser Ala Val Leu Glu 115 120 125 Lys Leu Lys Glu Tyr Ser Ser Met AspAla Phe Trp Gln Glu Gly Arg 130 135 140 Ala Ser Gly Thr Val Tyr Ser GlyGlu Glu Lys Leu Thr Glu Leu Leu 145 150 155 160 Val Lys Ala Tyr Gly AspPhe Ala Trp Ser Asn Pro Leu His Pro Asp 165 170 175 Ile Phe Pro Gly LeuArg Lys Ile Glu Ala Glu Ile Val Arg Ile Ala 180 185 190 Cys Ser Leu PheAsn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr Ser 195 200 205 Gly Gly ThrGlu Ser Ile Leu Met Ala Cys Lys Ala Cys Arg Asp Leu 210 215 220 Ala PheGlu Lys Gly Ile Lys Thr Pro Glu Ile Val Ala Pro Gln Ser 225 230 235 240Ala His Ala Ala Phe Asn Lys Ala Ala Ser Tyr Phe Gly Met Lys Ile 245 250255 Val Arg Val Pro Leu Thr Lys Met Met Glu Val Asp Val Arg Ala Met 260265 270 Arg Arg Ala Ile Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro275 280 285 Gln Phe Pro His Gly Val Ile Asp Pro Val Pro Glu Val Ala LysLeu 290 295 300 Ala Val Lys Tyr Lys Ile Pro Leu His Val Asp Ala Cys LeuGly Gly 305 310 315 320 Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr ProLeu Glu His Pro 325 330 335 Phe Asp Phe Arg Val Lys Gly Val Thr Ser IleSer Ala Asp Thr His 340 345 350 Lys Tyr Gly Tyr Ala Pro Lys Gly Ser SerLeu Val Leu Tyr Ser Asp 355 360 365 Lys Lys Tyr Arg Asn Tyr Gln Phe PheVal Asp Thr Asp Trp Gln Gly 370 375 380 Gly Ile Tyr Ala Ser Pro Thr IleAla Gly Ser Arg Pro Gly Gly Ile 385 390 395 400 Ser Ala Ala Cys Trp AlaAla Leu Met His Phe Gly Glu Asn Gly Tyr 405 410 415 Val Glu Ala Thr LysGln Ile Ile Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430 Glu Leu Glu AsnIle Lys Gly Ile Phe Val Phe Gly Asn Pro Gln Leu 435 440 445 Ser Leu IleAla Leu Gly Ser Arg Asp Phe Asp Ile Tyr Arg Leu Ser 450 455 460 Asn LeuMet Thr Ala Lys Gly Trp Asn Leu Asn Gln Leu Gln Phe Pro 465 470 475 480Pro Ser Ile His Phe Cys Ile Thr Leu Leu His Ala Arg Lys Arg Val 485 490495 Ala Ile Gln Phe Leu Lys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 500505 510 Lys Asn Pro Lys Ala Lys Thr Thr Gly Met Gly Ala Ile Tyr Ala Met515 520 525 Ala Gln Thr Thr Val Asp Arg Asn Met Val Ala Glu Leu Ser SerVal 530 535 540 Phe Leu Asp Ser Leu Tyr Ser Thr Asp Thr Val Thr Gln GlySer Gln 545 550 555 560 Met Asn Gly Ser Pro Lys Pro His 565 9 1467 DNAHomo sapiens CDS (1)...(1467) 9 atg cct agc aca gac ctt ctg atg ttg aaggcc ttt gag ccc tac tta 48 Met Pro Ser Thr Asp Leu Leu Met Leu Lys AlaPhe Glu Pro Tyr Leu 1 5 10 15 gag att ttg gaa gta tac tcc aca aaa gccaag aat tat gta aat gga 96 Glu Ile Leu Glu Val Tyr Ser Thr Lys Ala LysAsn Tyr Val Asn Gly 20 25 30 cat tgc acc aag tat gag ccc tgg cag cta attgca tgg agt gtc gtg 144 His Cys Thr Lys Tyr Glu Pro Trp Gln Leu Ile AlaTrp Ser Val Val 35 40 45 tgg acc ctg ctg ata gtc tgg gga tat gag ttt gtcttc cag cca gag 192 Trp Thr Leu Leu Ile Val Trp Gly Tyr Glu Phe Val PheGln Pro Glu 50 55 60 agt tta tgg tca agg ttt aaa aag aaa tgt ttt aag ctcacc agg aag 240 Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys Phe Lys Leu ThrArg Lys 65 70 75 80 atg ccc att att ggt cgt aag att caa gac aag ttg aacaag acc aag 288 Met Pro Ile Ile Gly Arg Lys Ile Gln Asp Lys Leu Asn LysThr Lys 85 90 95 gat gat att agc aag aac atg tca ttc ctg aaa gtg gac aaagag tat 336 Asp Asp Ile Ser Lys Asn Met Ser Phe Leu Lys Val Asp Lys GluTyr 100 105 110 gtg aaa gct tta ccc tcc cag ggt ctg agc tca tct gct gttttg gag 384 Val Lys Ala Leu Pro Ser Gln Gly Leu Ser Ser Ser Ala Val LeuGlu 115 120 125 aaa ctt aag gag tac agc tct atg gac gcc ttc tgg caa gagggg aga 432 Lys Leu Lys Glu Tyr Ser Ser Met Asp Ala Phe Trp Gln Glu GlyArg 130 135 140 gcc tct gga aca gtg tac agt ggg gag gag aag ctc act gagctc ctt 480 Ala Ser Gly Thr Val Tyr Ser Gly Glu Glu Lys Leu Thr Glu LeuLeu 145 150 155 160 gtg aag gct tat gga gat ttt gca tgg agt aac ccc ctgcat cca gat 528 Val Lys Ala Tyr Gly Asp Phe Ala Trp Ser Asn Pro Leu HisPro Asp 165 170 175 atc ttc cca gga cta cgc aag ata gag gca gaa att gtgagg ata gct 576 Ile Phe Pro Gly Leu Arg Lys Ile Glu Ala Glu Ile Val ArgIle Ala 180 185 190 tgt tcc ctg ttc aat ggg gga cca gat tcg tgt gga tgtgtg act tct 624 Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys ValThr Ser 195 200 205 ggg gga aca gaa agc ata ctc atg gcc tgc aaa gca tgtcgg gat ctg 672 Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys Ala Cys ArgAsp Leu 210 215 220 gcc ttt gag aag ggg atc aaa act cca gaa att gtg gctccc caa agt 720 Ala Phe Glu Lys Gly Ile Lys Thr Pro Glu Ile Val Ala ProGln Ser 225 230 235 240 gcc cat gct gca ttt aac aaa gca gcc agt tac tttggg atg aag att 768 Ala His Ala Ala Phe Asn Lys Ala Ala Ser Tyr Phe GlyMet Lys Ile 245 250 255 gtg cgg gtc cca ttg acg aag atg atg gag gtg gatgtg agg gca atg 816 Val Arg Val Pro Leu Thr Lys Met Met Glu Val Asp ValArg Ala Met 260 265 270 aga aga gct atc tcc agg aac act gcc atg ctc gtctgt tct acc cca 864 Arg Arg Ala Ile Ser Arg Asn Thr Ala Met Leu Val CysSer Thr Pro 275 280 285 cag ttt cct cat ggt gta ata gat cct gtc cct gaagtg gcc aag ctg 912 Gln Phe Pro His Gly Val Ile Asp Pro Val Pro Glu ValAla Lys Leu 290 295 300 gct gtc aaa tac aaa ata ccc ctt cat gtc gac gcttgt ctg gga ggc 960 Ala Val Lys Tyr Lys Ile Pro Leu His Val Asp Ala CysLeu Gly Gly 305 310 315 320 ttc ctc atc gtc ttt atg gag aaa gca gga taccca ctg gag cac cca 1008 Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr ProLeu Glu His Pro 325 330 335 ttt gat ttc cgg gtg aaa ggt gta acc agc atttca gct gac acc cat 1056 Phe Asp Phe Arg Val Lys Gly Val Thr Ser Ile SerAla Asp Thr His 340 345 350 aag ctg gaa aat atc aaa ggc atc ttt gtt tttggg aat ccc caa ttg 1104 Lys Leu Glu Asn Ile Lys Gly Ile Phe Val Phe GlyAsn Pro Gln Leu 355 360 365 tca ctc att gct ctg gga tcc cgt gat ttt gacatc tac cga cta tca 1152 Ser Leu Ile Ala Leu Gly Ser Arg Asp Phe Asp IleTyr Arg Leu Ser 370 375 380 aac ctg atg act gct aag ggg tgg aac ttg aaccag ttg cag ttc cca 1200 Asn Leu Met Thr Ala Lys Gly Trp Asn Leu Asn GlnLeu Gln Phe Pro 385 390 395 400 ccc agt att cat ttc tgc atc aca tta ctacac gcc cgg aaa cga gta 1248 Pro Ser Ile His Phe Cys Ile Thr Leu Leu HisAla Arg Lys Arg Val 405 410 415 gct ata caa ttc cta aag gac att cga gaatct gtc act caa atc atg 1296 Ala Ile Gln Phe Leu Lys Asp Ile Arg Glu SerVal Thr Gln Ile Met 420 425 430 aag aat cct aaa gcg aag acc aca gga atgggt gcc atc tat gcc atg 1344 Lys Asn Pro Lys Ala Lys Thr Thr Gly Met GlyAla Ile Tyr Ala Met 435 440 445 gcc cag aca act gtt gac agg aat atg gttgca gaa ttg tcc tca gtc 1392 Ala Gln Thr Thr Val Asp Arg Asn Met Val AlaGlu Leu Ser Ser Val 450 455 460 ttc ttg gac agc ttg tac agc acc gac actgtc acc cag ggc agc cag 1440 Phe Leu Asp Ser Leu Tyr Ser Thr Asp Thr ValThr Gln Gly Ser Gln 465 470 475 480 atg aat ggt tct cca aaa ccc cac tga1467 Met Asn Gly Ser Pro Lys Pro His * 485 10 488 PRT Homo sapiens 10Met Pro Ser Thr Asp Leu Leu Met Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 1015 Glu Ile Leu Glu Val Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 2530 His Cys Thr Lys Tyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val Val 35 4045 Trp Thr Leu Leu Ile Val Trp Gly Tyr Glu Phe Val Phe Gln Pro Glu 50 5560 Ser Leu Trp Ser Arg Phe Lys Lys Lys Cys Phe Lys Leu Thr Arg Lys 65 7075 80 Met Pro Ile Ile Gly Arg Lys Ile Gln Asp Lys Leu Asn Lys Thr Lys 8590 95 Asp Asp Ile Ser Lys Asn Met Ser Phe Leu Lys Val Asp Lys Glu Tyr100 105 110 Val Lys Ala Leu Pro Ser Gln Gly Leu Ser Ser Ser Ala Val LeuGlu 115 120 125 Lys Leu Lys Glu Tyr Ser Ser Met Asp Ala Phe Trp Gln GluGly Arg 130 135 140 Ala Ser Gly Thr Val Tyr Ser Gly Glu Glu Lys Leu ThrGlu Leu Leu 145 150 155 160 Val Lys Ala Tyr Gly Asp Phe Ala Trp Ser AsnPro Leu His Pro Asp 165 170 175 Ile Phe Pro Gly Leu Arg Lys Ile Glu AlaGlu Ile Val Arg Ile Ala 180 185 190 Cys Ser Leu Phe Asn Gly Gly Pro AspSer Cys Gly Cys Val Thr Ser 195 200 205 Gly Gly Thr Glu Ser Ile Leu MetAla Cys Lys Ala Cys Arg Asp Leu 210 215 220 Ala Phe Glu Lys Gly Ile LysThr Pro Glu Ile Val Ala Pro Gln Ser 225 230 235 240 Ala His Ala Ala PheAsn Lys Ala Ala Ser Tyr Phe Gly Met Lys Ile 245 250 255 Val Arg Val ProLeu Thr Lys Met Met Glu Val Asp Val Arg Ala Met 260 265 270 Arg Arg AlaIle Ser Arg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275 280 285 Gln PhePro His Gly Val Ile Asp Pro Val Pro Glu Val Ala Lys Leu 290 295 300 AlaVal Lys Tyr Lys Ile Pro Leu His Val Asp Ala Cys Leu Gly Gly 305 310 315320 Phe Leu Ile Val Phe Met Glu Lys Ala Gly Tyr Pro Leu Glu His Pro 325330 335 Phe Asp Phe Arg Val Lys Gly Val Thr Ser Ile Ser Ala Asp Thr His340 345 350 Lys Leu Glu Asn Ile Lys Gly Ile Phe Val Phe Gly Asn Pro GlnLeu 355 360 365 Ser Leu Ile Ala Leu Gly Ser Arg Asp Phe Asp Ile Tyr ArgLeu Ser 370 375 380 Asn Leu Met Thr Ala Lys Gly Trp Asn Leu Asn Gln LeuGln Phe Pro 385 390 395 400 Pro Ser Ile His Phe Cys Ile Thr Leu Leu HisAla Arg Lys Arg Val 405 410 415 Ala Ile Gln Phe Leu Lys Asp Ile Arg GluSer Val Thr Gln Ile Met 420 425 430 Lys Asn Pro Lys Ala Lys Thr Thr GlyMet Gly Ala Ile Tyr Ala Met 435 440 445 Ala Gln Thr Thr Val Asp Arg AsnMet Val Ala Glu Leu Ser Ser Val 450 455 460 Phe Leu Asp Ser Leu Tyr SerThr Asp Thr Val Thr Gln Gly Ser Gln 465 470 475 480 Met Asn Gly Ser ProLys Pro His 485 11 552 PRT C. elegans 11 Met Asp Ser Val Lys His Thr ThrGlu Ile Ile Val Asp Leu Thr Lys 1 5 10 15 Met His Tyr His Met Ile AsnAsp Arg Leu Ser Arg Tyr Asp Pro Val 20 25 30 Val Leu Val Leu Ala Ala PheGly Gly Thr Leu Val Tyr Thr Lys Val 35 40 45 Val His Leu Tyr Arg Lys SerGlu Asp Pro Ile Leu Lys Arg Met Gly 50 55 60 Ala Tyr Val Phe Ser Leu LeuArg Lys Leu Pro Ala Val Arg Asp Lys 65 70 75 80 Ile Glu Lys Glu Leu AlaAla Glu Lys Pro Lys Leu Ile Glu Ser Ile 85 90 95 His Lys Asp Asp Lys AspLys Gln Phe Ile Ser Thr Leu Pro Ile Ala 100 105 110 Pro Leu Ser Gln AspSer Ile Met Glu Leu Ala Lys Lys Tyr Glu Asp 115 120 125 Tyr Asn Thr PheAsn Ile Asp Gly Gly Arg Val Ser Gly Ala Val Tyr 130 135 140 Thr Asp ArgHis Ala Glu His Ile Asn Leu Leu Gly Lys Ile Tyr Glu 145 150 155 160 LysTyr Ala Phe Ser Asn Pro Leu His Pro Asp Val Phe Pro Gly Ala 165 170 175Arg Lys Met Glu Ala Glu Leu Ile Arg Met Val Leu Asn Leu Tyr Asn 180 185190 Gly Pro Glu Asp Ser Ser Gly Ser Val Thr Ser Gly Gly Thr Glu Ser 195200 205 Ile Ile Met Ala Cys Phe Ser Tyr Arg Asn Arg Ala His Ser Leu Gly210 215 220 Ile Glu His Pro Val Ile Leu Ala Cys Lys Thr Ala His Ala AlaPhe 225 230 235 240 Asp Lys Ala Ala His Leu Cys Gly Met Arg Leu Arg HisVal Pro Val 245 250 255 Asp Ser Asp Asn Arg Val Asp Leu Lys Glu Met GluArg Leu Ile Asp 260 265 270 Ser Asn Val Cys Met Leu Val Gly Ser Ala ProAsn Phe Pro Ser Gly 275 280 285 Thr Ile Asp Pro Ile Pro Glu Ile Ala LysLeu Gly Lys Lys Tyr Gly 290 295 300 Ile Pro Val His Val Asp Ala Cys LeuGly Gly Phe Met Ile Pro Phe 305 310 315 320 Met Asn Asp Ala Gly Tyr LeuIle Pro Val Phe Asp Phe Arg Asn Pro 325 330 335 Gly Val Thr Ser Ile SerCys Asp Thr His Lys Tyr Gly Cys Thr Pro 340 345 350 Lys Gly Ser Ser IleVal Met Tyr Arg Ser Lys Glu Leu His His Phe 355 360 365 Gln Tyr Phe SerVal Ala Asp Trp Cys Gly Gly Ile Tyr Ala Thr Pro 370 375 380 Thr Ile AlaGly Ser Arg Ala Gly Ala Asn Thr Ala Val Ala Trp Ala 385 390 395 400 ThrLeu Leu Ser Phe Gly Arg Asp Glu Tyr Val Arg Arg Cys Ala Gln 405 410 415Ile Val Lys His Thr Arg Met Leu Ala Glu Lys Ile Glu Lys Ile Lys 420 425430 Trp Ile Lys Pro Tyr Gly Lys Ser Asp Val Ser Leu Val Ala Phe Ser 435440 445 Gly Asn Gly Val Asn Ile Tyr Glu Val Ser Asp Lys Met Met Lys Leu450 455 460 Gly Trp Asn Leu Asn Thr Leu Gln Asn Pro Ala Ala Ile His IleCys 465 470 475 480 Leu Thr Ile Asn Gln Ala Asn Glu Glu Val Val Asn AlaPhe Ala Val 485 490 495 Asp Leu Glu Lys Ile Cys Glu Glu Leu Ala Ala LysGly Glu Gln Lys 500 505 510 Ala Asp Ser Gly Met Ala Ala Met Tyr Gly MetAla Ala Gln Val Pro 515 520 525 Lys Ser Val Val Asp Glu Val Ile Ala LeuTyr Ile Asp Ala Thr Tyr 530 535 540 Ser Ala Pro Pro Ser Thr Ser Asn 545550 12 3162 DNA C. elegans 12 atggattcgg ttaagcacac aaccgaaattattgtcgact tgacaaaaat gcactatcac 60 atgataaatg ataggtgaat tttaaacaaaaattagatat ttggaaatta ctaattcaag 120 attttcagac tttctcggta tgatccggttgttctagtgt tggccgcttt tgggggtacc 180 cttgtctata caaaagtcgt ccatttgtaccgaaaaagcg aggatccaat tttgaaacgg 240 caagtgtttt cttgcgaatt ttagaaatatcaaaatgaaa ttttcagcat gggagcttat 300 gtattctcac ttcttcgaaa acttccagctgttcgggata aaatcgaaaa agagctggct 360 gctgagaagc caaagcttat tgaatcgattcataaggatg ataaggacaa gcaattcatt 420 tccagtttgt ttgaacattt attaattaaccaattcatta attctatttt tcagctcttc 480 ccatcgctcc attatctcag gactcaattatggaactggc gaaaaaatat gaggattaca 540 acacatttaa cattgacgga ggacgagtatctggagcggt ttatactgat cgtcatgctg 600 aacacattaa tttgcttgga aaggtttagaaattctagaa tttttcaaaa tcttagctct 660 caaatatatt ctcttgtaaa tagctacatagtatatcctg tagggaagct ttgaatccaa 720 ttcagatcag gggcgacaaa cgattttttccggcaaatcg gcaaatcgcc ggaatggaaa 780 tttcctgcaa atcggcaaat tgccggaatggaaatttcct gcaagttggc aaattgacgg 840 aattgaaatt tccggcaaac cgacaaatttccgtaattaa aatttcctgc aaaccggcga 900 attggcggaa ttgaaatttc ctgcaaaccggcaaattgcc gtaattgaaa tttcctgcaa 960 accggcaaat tgccggaatt gaaatttccggcaaaccggc aaatcggctg aattgaaatt 1020 tcctgcaaac cggcaaattg cggtaattgaaatttcctgc aaaccggtca gttgccgatt 1080 tgcctttgcc tgaaaaacgg cgattgccagaaatattcgg caaattgtgg ttttgcacat 1140 ttttctggaa atttcaggca aaattgtacgcatcctatga atatccctat taacatcttt 1200 tttgaaaagt cagtaaatta tatgaaaatatctaaagaaa acggggaaaa tatttcaaag 1260 aggcacagtt ttatgtgttt ccgtcatctaaatagtccct ctaaacattt ccggcaaatc 1320 tgatatccgg caaacggcaa atcgggatattgccggaatt taaaatttgc cgaacttgtc 1380 gacaaaaaaa atgcgccttg aatccgattcagatattcaa aaattgaatt ttggacgttt 1440 tagaaatcat ttagtttgtc aattttcaagaaatttctag aaaattggat ggtttccgcc 1500 aagaaatatt agctacatga aaataattttgaaactagac atttcttaaa ataaaaattg 1560 ccatctttta tatccagatt tacgaaaagtatgcgttctc gaatcccctc caccctgacg 1620 tctttccggg agctcgtaaa atggaggcagaacttattcg aatggttctg aacctgtata 1680 atggaccaga agattctagt ggaagtgtaacttctggtgg tactgaaagt attattatgg 1740 catgcttttc gtatcggtaa gcatttattcaactcttaaa attcaatttt gcaaactcta 1800 cagaaatcgt gcacactctc ttggcattgaacatccagtt attttggcat gtaaaacagc 1860 tcacgcggca tttgataagg ccgcccatctatgcggaatg cgtcttcgcc acgttccagt 1920 tgattcggat aatcgtgtcg atttaaaagaaatggagaga ctaattgatt cgaatgtttg 1980 tatgttggtt ggctcagcgc ctaacttcccatcaggcaca attgatccaa ttccggaaat 2040 tgctaaggta ctggaaattc ccgcctcaatatcgcggaaa aaatagagaa atgactgaac 2100 aaaattacat tgtgagcggg aactctaattgaattcagca aaaatacgat acttttttct 2160 aacttaaaat aatttttaaa aaaactcacagatgctagtc caaaaaatgg ccttttttga 2220 ttacttaatc gaacgtttac actttcagctcggcaaaaag tatggaatcc cggtccacgt 2280 ggacgcatgt cttggtggat tcatgattccatttatgaat gacgccggat acctgattcc 2340 tgtattcgat ttcagaaatc ccggtgttacatctatttcg tgtgatactc ataaggttgg 2400 atacagttct atccattttt ttccttcaattcaaaatctt tcagtacgga tgcacaccga 2460 aaggttcatc gattgtcatg tatcgttccaaggaacttca tcacttccag tatttctcgg 2520 ttgccgattg gtgtggaggc atctatgccaccccgactat tgcaggtttg aagaatgttt 2580 tagtagcttc aatagaatca aagagatcccttaggatccc gagctggagc caacactgcc 2640 gtcgcctggg ccacactttt atccttcggtcgagacgaat atgttcgaag atgtgctcaa 2700 attgtgaagc atacacgaat gctggccgagaaaattgaga aaatcaaatg gatcaagcct 2760 tatggaaaat cggatgtttc attggtggcgttctccggaa atggtgtgaa tatctacgaa 2820 gtttctgaca aaatgatgaa gctcggatggaatttgaaca ctctgcagaa tccagcggcg 2880 tatgtttatc aattttatga gttatcagcttgctaaattt tttgtttcag aatccacatt 2940 tgtttgacaa tcaatcaagc gaacgaggaagttgtgaatg cgttcgccgt cgaccttgag 3000 aagatttgtg aagaactcgc tgcaaaaggtgaacaaaaag ctgacagtgg aatggctgcg 3060 atgtatggaa tggctgcgca agtaccaaaatcagtagtgg acgaggttat cgctctgtac 3120 attgacgcaa cttattcagc tccaccttcaacttctaatt aa 3162 13 34 DNA Artificial Sequence primer 13 gaggaattcatggattcggt taagcacaca accg 34 14 33 DNA Artificial Sequence primer 14agcctcgagt taattagaag ttgaaggtgg agc 33 15 1638 DNA Drosophilamelanogaster 15 atgcgtccgt tctccggcag cgattgcctt aagcccgtca ccgagggcatcaaccgggcg 60 ttcggcgcca aggagccctg gcaggtggcc accatcacgg ccaccacggtgctgggaggc 120 gtctggctct ggactgtgat ctgccaggat gaaaatcttt acattcgtggcaagcgtcag 180 ttctttaagt ttgccaagaa gattccagcc gtgcgtcgtc aggtggagactgaattggcc 240 aaggccaaaa acgacttcga gacggaaatc aaaaagagca acgcccaccttacctactcg 300 gaaactctgc ccgagaaggg actcagcaag gaggagatcc tccgactggtggatgagcac 360 ctgaagactg gtcactacaa ctggcgtgat ggtcgtgtat ctggcgcggtctacggctac 420 aagcctgatc tggtggagct cgtcactgaa gtgtacggca aggcctcctacaccaatccc 480 ttgcacgcag atcttttccc gggagtttgc aaaatggagg cggaggtagtgcgcatggca 540 tgcaacctgt tccatggaaa ctcagccagc tgtggaacca tgaccaccggcggcaccgaa 600 tccattgtaa tggccatgaa ggcgtacagg gatttcgcta gagagtacaagggaatcacc 660 aggccaaaca tcgtggtgcc taagacggtc cacgcggcct tcgacaagggcggtcagtac 720 tttaatatcc acgtgcgatc cgtggatgta gatccggaga cctacgaagtggacattaag 780 aagttcaaac gtgccattaa caggaacacg attctgctgg ttgggtctgctccgaacttc 840 ccctatggaa ccatcgatga catcgaagct atcgccgctt tgggcgttaagtacgacatt 900 cccgtgcacg tggacgcctg cctgggcagc tttgtggtgg ccttggtccgcaacgccggc 960 tataagctgc gtcccttcga ctttgaggtc aagggagtga ccagtatctccgctgatacc 1020 cacaagtatg gtttcgcgcc caagggatca tcggtgatcc tttactcggacaagaagtac 1080 aaggaccatc agttcactgt gactactgac tggcctggcg gcgtgtatggttctcccaca 1140 gtcaacggtt cccgtgccgg aggtattatc gccgcctgct gggctaccatgatgagcttt 1200 ggctatgatg gttatctgga agccactaag cgcattgtgg atacggcgcgctatatcgag 1260 aggggcgttc gcgacatcga tggcatcttt atctttggca agccagctacttcagtgatt 1320 gccctgggtt ccaatgtgtt tgacattttc cggctatcgg attcgctgtgcaaactgggc 1380 tggaacctaa atgcgctgca gtttccatct ggtatccacc tgtgcgtgacggacatgcac 1440 acacagcccg gagtcgcgga taaattcatt gccgatgtgc gcagctgtacggcggagatc 1500 atgaaggatc ccggccagcc cgtcgttgga aagatggctc tttacggcatggcacagagc 1560 atacccgacc gttcggtgat cggagaagtg actcgcctat tcctgcactccatgtactac 1620 actcccagcc agaaatag 1638 16 545 PRT Drosophilamelanogaster 16 Met Arg Pro Phe Ser Gly Ser Asp Cys Leu Lys Pro Val ThrGlu Gly 1 5 10 15 Ile Asn Arg Ala Phe Gly Ala Lys Glu Pro Trp Gln ValAla Thr Ile 20 25 30 Thr Ala Thr Thr Val Leu Gly Gly Val Trp Leu Trp ThrVal Ile Cys 35 40 45 Gln Asp Glu Asn Leu Tyr Ile Arg Gly Lys Arg Gln PhePhe Lys Phe 50 55 60 Ala Lys Lys Ile Pro Ala Val Arg Arg Gln Val Glu ThrGlu Leu Ala 65 70 75 80 Lys Ala Lys Asn Asp Phe Glu Thr Glu Ile Lys LysSer Asn Ala His 85 90 95 Leu Thr Tyr Ser Glu Thr Leu Pro Glu Lys Gly LeuSer Lys Glu Glu 100 105 110 Ile Leu Arg Leu Val Asp Glu His Leu Lys ThrGly His Tyr Asn Trp 115 120 125 Arg Asp Gly Arg Val Ser Gly Ala Val TyrGly Tyr Lys Pro Asp Leu 130 135 140 Val Glu Leu Val Thr Glu Val Tyr GlyLys Ala Ser Tyr Thr Asn Pro 145 150 155 160 Leu His Ala Asp Leu Phe ProGly Val Cys Lys Met Glu Ala Glu Val 165 170 175 Val Arg Met Ala Cys AsnLeu Phe His Gly Asn Ser Ala Ser Cys Gly 180 185 190 Thr Met Thr Thr GlyGly Thr Glu Ser Ile Val Met Ala Met Lys Ala 195 200 205 Tyr Arg Asp PheAla Arg Glu Tyr Lys Gly Ile Thr Arg Pro Asn Ile 210 215 220 Val Val ProLys Thr Val His Ala Ala Phe Asp Lys Gly Gly Gln Tyr 225 230 235 240 PheAsn Ile His Val Arg Ser Val Asp Val Asp Pro Glu Thr Tyr Glu 245 250 255Val Asp Ile Lys Lys Phe Lys Arg Ala Ile Asn Arg Asn Thr Ile Leu 260 265270 Leu Val Gly Ser Ala Pro Asn Phe Pro Tyr Gly Thr Ile Asp Asp Ile 275280 285 Glu Ala Ile Ala Ala Leu Gly Val Lys Tyr Asp Ile Pro Val His Val290 295 300 Asp Ala Cys Leu Gly Ser Phe Val Val Ala Leu Val Arg Asn AlaGly 305 310 315 320 Tyr Lys Leu Arg Pro Phe Asp Phe Glu Val Lys Gly ValThr Ser Ile 325 330 335 Ser Ala Asp Thr His Lys Tyr Gly Phe Ala Pro LysGly Ser Ser Val 340 345 350 Ile Leu Tyr Ser Asp Lys Lys Tyr Lys Asp HisGln Phe Thr Val Thr 355 360 365 Thr Asp Trp Pro Gly Gly Val Tyr Gly SerPro Thr Val Asn Gly Ser 370 375 380 Arg Ala Gly Gly Ile Ile Ala Ala CysTrp Ala Thr Met Met Ser Phe 385 390 395 400 Gly Tyr Asp Gly Tyr Leu GluAla Thr Lys Arg Ile Val Asp Thr Ala 405 410 415 Arg Tyr Ile Glu Arg GlyVal Arg Asp Ile Asp Gly Ile Phe Ile Phe 420 425 430 Gly Lys Pro Ala ThrSer Val Ile Ala Leu Gly Ser Asn Val Phe Asp 435 440 445 Ile Phe Arg LeuSer Asp Ser Leu Cys Lys Leu Gly Trp Asn Leu Asn 450 455 460 Ala Leu GlnPhe Pro Ser Gly Ile His Leu Cys Val Thr Asp Met His 465 470 475 480 ThrGln Pro Gly Val Ala Asp Lys Phe Ile Ala Asp Val Arg Ser Cys 485 490 495Thr Ala Glu Ile Met Lys Asp Pro Gly Gln Pro Val Val Gly Lys Met 500 505510 Ala Leu Tyr Gly Met Ala Gln Ser Ile Pro Asp Arg Ser Val Ile Gly 515520 525 Glu Val Thr Arg Leu Phe Leu His Ser Met Tyr Tyr Thr Pro Ser Gln530 535 540 Lys 545 17 1707 DNA Homo sapiens CDS (1)...(1707) 17 atg cctagc aca gac ctt ctg atg ttg aag gcc ttt gag ccc tac tta 48 Met Pro SerThr Asp Leu Leu Met Leu Lys Ala Phe Glu Pro Tyr Leu 1 5 10 15 gag attttg gaa gta tac tcc aca aaa gcc aag aat tat gta aat gga 96 Glu Ile LeuGlu Val Tyr Ser Thr Lys Ala Lys Asn Tyr Val Asn Gly 20 25 30 cat tgc accaag tat gag ccc tgg cag cta att gca tgg agt gtc gtg 144 His Cys Thr LysTyr Glu Pro Trp Gln Leu Ile Ala Trp Ser Val Val 35 40 45 tgg acc ctg ctgata gtc tgg gga tat gag ttt gtc ttc cag cca gag 192 Trp Thr Leu Leu IleVal Trp Gly Tyr Glu Phe Val Phe Gln Pro Glu 50 55 60 agt tta tgg tca aggttt aaa aag aaa tgt ttt aag ctc acc agg aag 240 Ser Leu Trp Ser Arg PheLys Lys Lys Cys Phe Lys Leu Thr Arg Lys 65 70 75 80 atg ccc att att ggtcgt aag att caa gac aag ttg aac aag acc aag 288 Met Pro Ile Ile Gly ArgLys Ile Gln Asp Lys Leu Asn Lys Thr Lys 85 90 95 gat gat att agc aag aacatg tca ttc ctg aaa gtg gac aaa gag tat 336 Asp Asp Ile Ser Lys Asn MetSer Phe Leu Lys Val Asp Lys Glu Tyr 100 105 110 gtg aaa gct tta ccc tcccag ggt ctg agc tca tct gct gtt ttg gag 384 Val Lys Ala Leu Pro Ser GlnGly Leu Ser Ser Ser Ala Val Leu Glu 115 120 125 aaa ctt aag gag tac agctct atg gac gcc ttc tgg caa gag ggg aga 432 Lys Leu Lys Glu Tyr Ser SerMet Asp Ala Phe Trp Gln Glu Gly Arg 130 135 140 gcc tct gga aca gtg tacagt ggg gag gag aag ctc act gag ctc ctt 480 Ala Ser Gly Thr Val Tyr SerGly Glu Glu Lys Leu Thr Glu Leu Leu 145 150 155 160 gtg aag gct tat ggagat ttt gca tgg agt aac ccc ctg cat cca gat 528 Val Lys Ala Tyr Gly AspPhe Ala Trp Ser Asn Pro Leu His Pro Asp 165 170 175 atc ttc cca gga ctacgc aag ata gag gca gaa att gtg agg ata gct 576 Ile Phe Pro Gly Leu ArgLys Ile Glu Ala Glu Ile Val Arg Ile Ala 180 185 190 tgt tcc ctg ttc aatggg gga cca gat tcg tgt gga tgt gtg act tct 624 Cys Ser Leu Phe Asn GlyGly Pro Asp Ser Cys Gly Cys Val Thr Ser 195 200 205 ggg gga aca gaa agcata ctc atg gcc tgc aaa gca tat cgg gat ctg 672 Gly Gly Thr Glu Ser IleLeu Met Ala Cys Lys Ala Tyr Arg Asp Leu 210 215 220 gcc ttt gag aag gggatc aaa act cca gaa att gtg gct ccc caa agt 720 Ala Phe Glu Lys Gly IleLys Thr Pro Glu Ile Val Ala Pro Gln Ser 225 230 235 240 gcc cat gct gcattt aac aaa gca gcc agt tac ttt ggg atg aag att 768 Ala His Ala Ala PheAsn Lys Ala Ala Ser Tyr Phe Gly Met Lys Ile 245 250 255 gtg cgg gtc ccattg acg aag atg atg gag gtg gat gtg agg gca atg 816 Val Arg Val Pro LeuThr Lys Met Met Glu Val Asp Val Arg Ala Met 260 265 270 aga aga gct atctcc agg aac act gcc atg ctc gtc tgt tct acc cca 864 Arg Arg Ala Ile SerArg Asn Thr Ala Met Leu Val Cys Ser Thr Pro 275 280 285 cag ttt cct catggt gta ata gat cct gtc cct gaa gtg gcc aag ctg 912 Gln Phe Pro His GlyVal Ile Asp Pro Val Pro Glu Val Ala Lys Leu 290 295 300 gct gtc aaa tacaaa ata ccc ctt cat gtc gac gct tgt ctg gga ggc 960 Ala Val Lys Tyr LysIle Pro Leu His Val Asp Ala Cys Leu Gly Gly 305 310 315 320 ttc ctc atcgtc ttt atg gag aaa gca gga tac cca ctg gag cac cca 1008 Phe Leu Ile ValPhe Met Glu Lys Ala Gly Tyr Pro Leu Glu His Pro 325 330 335 ttt gat ttccgg gtg aaa ggt gta acc agc att tca gct gac acc cat 1056 Phe Asp Phe ArgVal Lys Gly Val Thr Ser Ile Ser Ala Asp Thr His 340 345 350 aag tat ggctat gcc cca aaa ggc tca tca ttg gtg ttg tat agt gac 1104 Lys Tyr Gly TyrAla Pro Lys Gly Ser Ser Leu Val Leu Tyr Ser Asp 355 360 365 aag aag tacagg aac tat cag ttc ttc gtc gat aca gat tgg cag ggt 1152 Lys Lys Tyr ArgAsn Tyr Gln Phe Phe Val Asp Thr Asp Trp Gln Gly 370 375 380 ggc atc tatgct tcc cca acc atc gca ggc tca cgg cct ggt ggc att 1200 Gly Ile Tyr AlaSer Pro Thr Ile Ala Gly Ser Arg Pro Gly Gly Ile 385 390 395 400 agc gcagcc tgt tgg gct gcc ttg atg cac ttc ggt gag aac ggc tat 1248 Ser Ala AlaCys Trp Ala Ala Leu Met His Phe Gly Glu Asn Gly Tyr 405 410 415 gtt gaagct acc aaa cag atc atc aaa act gct cgc ttc ctc aag tca 1296 Val Glu AlaThr Lys Gln Ile Ile Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430 gaa ctggaa aat atc aaa ggc atc ttt gtt ttt ggg aat ccc caa ttg 1344 Glu Leu GluAsn Ile Lys Gly Ile Phe Val Phe Gly Asn Pro Gln Leu 435 440 445 tca gtcatt gct ctg gga tcc cgt gat ttt gac atc tac cga cta tca 1392 Ser Val IleAla Leu Gly Ser Arg Asp Phe Asp Ile Tyr Arg Leu Ser 450 455 460 aac ctgatg act gct aag ggg tgg aac ttg aac cag ttg cag ttc cca 1440 Asn Leu MetThr Ala Lys Gly Trp Asn Leu Asn Gln Leu Gln Phe Pro 465 470 475 480 cccagt att cat ttc tgc atc aca tta cta cac gcc cgg aaa cga gta 1488 Pro SerIle His Phe Cys Ile Thr Leu Leu His Ala Arg Lys Arg Val 485 490 495 gctata caa ttc cta aag gac att cga gaa tct gtc act caa atc atg 1536 Ala IleGln Phe Leu Lys Asp Ile Arg Glu Ser Val Thr Gln Ile Met 500 505 510 aagaat cct aaa gcg aag acc aca gga atg ggt gcc atc tat ggc atg 1584 Lys AsnPro Lys Ala Lys Thr Thr Gly Met Gly Ala Ile Tyr Gly Met 515 520 525 gcccag aca act gtt gac agg aat atg gtt gca gaa ttg tcc tca gtc 1632 Ala GlnThr Thr Val Asp Arg Asn Met Val Ala Glu Leu Ser Ser Val 530 535 540 ttcttg gac agc ttg tac agc acc gac act gtc acc cag ggc agc cag 1680 Phe LeuAsp Ser Leu Tyr Ser Thr Asp Thr Val Thr Gln Gly Ser Gln 545 550 555 560atg aat ggt tct cca aaa ccc cac tga 1707 Met Asn Gly Ser Pro Lys ProHis * 565 18 568 PRT Homo sapiens 18 Met Pro Ser Thr Asp Leu Leu Met LeuLys Ala Phe Glu Pro Tyr Leu 1 5 10 15 Glu Ile Leu Glu Val Tyr Ser ThrLys Ala Lys Asn Tyr Val Asn Gly 20 25 30 His Cys Thr Lys Tyr Glu Pro TrpGln Leu Ile Ala Trp Ser Val Val 35 40 45 Trp Thr Leu Leu Ile Val Trp GlyTyr Glu Phe Val Phe Gln Pro Glu 50 55 60 Ser Leu Trp Ser Arg Phe Lys LysLys Cys Phe Lys Leu Thr Arg Lys 65 70 75 80 Met Pro Ile Ile Gly Arg LysIle Gln Asp Lys Leu Asn Lys Thr Lys 85 90 95 Asp Asp Ile Ser Lys Asn MetSer Phe Leu Lys Val Asp Lys Glu Tyr 100 105 110 Val Lys Ala Leu Pro SerGln Gly Leu Ser Ser Ser Ala Val Leu Glu 115 120 125 Lys Leu Lys Glu TyrSer Ser Met Asp Ala Phe Trp Gln Glu Gly Arg 130 135 140 Ala Ser Gly ThrVal Tyr Ser Gly Glu Glu Lys Leu Thr Glu Leu Leu 145 150 155 160 Val LysAla Tyr Gly Asp Phe Ala Trp Ser Asn Pro Leu His Pro Asp 165 170 175 IlePhe Pro Gly Leu Arg Lys Ile Glu Ala Glu Ile Val Arg Ile Ala 180 185 190Cys Ser Leu Phe Asn Gly Gly Pro Asp Ser Cys Gly Cys Val Thr Ser 195 200205 Gly Gly Thr Glu Ser Ile Leu Met Ala Cys Lys Ala Tyr Arg Asp Leu 210215 220 Ala Phe Glu Lys Gly Ile Lys Thr Pro Glu Ile Val Ala Pro Gln Ser225 230 235 240 Ala His Ala Ala Phe Asn Lys Ala Ala Ser Tyr Phe Gly MetLys Ile 245 250 255 Val Arg Val Pro Leu Thr Lys Met Met Glu Val Asp ValArg Ala Met 260 265 270 Arg Arg Ala Ile Ser Arg Asn Thr Ala Met Leu ValCys Ser Thr Pro 275 280 285 Gln Phe Pro His Gly Val Ile Asp Pro Val ProGlu Val Ala Lys Leu 290 295 300 Ala Val Lys Tyr Lys Ile Pro Leu His ValAsp Ala Cys Leu Gly Gly 305 310 315 320 Phe Leu Ile Val Phe Met Glu LysAla Gly Tyr Pro Leu Glu His Pro 325 330 335 Phe Asp Phe Arg Val Lys GlyVal Thr Ser Ile Ser Ala Asp Thr His 340 345 350 Lys Tyr Gly Tyr Ala ProLys Gly Ser Ser Leu Val Leu Tyr Ser Asp 355 360 365 Lys Lys Tyr Arg AsnTyr Gln Phe Phe Val Asp Thr Asp Trp Gln Gly 370 375 380 Gly Ile Tyr AlaSer Pro Thr Ile Ala Gly Ser Arg Pro Gly Gly Ile 385 390 395 400 Ser AlaAla Cys Trp Ala Ala Leu Met His Phe Gly Glu Asn Gly Tyr 405 410 415 ValGlu Ala Thr Lys Gln Ile Ile Lys Thr Ala Arg Phe Leu Lys Ser 420 425 430Glu Leu Glu Asn Ile Lys Gly Ile Phe Val Phe Gly Asn Pro Gln Leu 435 440445 Ser Val Ile Ala Leu Gly Ser Arg Asp Phe Asp Ile Tyr Arg Leu Ser 450455 460 Asn Leu Met Thr Ala Lys Gly Trp Asn Leu Asn Gln Leu Gln Phe Pro465 470 475 480 Pro Ser Ile His Phe Cys Ile Thr Leu Leu His Ala Arg LysArg Val 485 490 495 Ala Ile Gln Phe Leu Lys Asp Ile Arg Glu Ser Val ThrGln Ile Met 500 505 510 Lys Asn Pro Lys Ala Lys Thr Thr Gly Met Gly AlaIle Tyr Gly Met 515 520 525 Ala Gln Thr Thr Val Asp Arg Asn Met Val AlaGlu Leu Ser Ser Val 530 535 540 Phe Leu Asp Ser Leu Tyr Ser Thr Asp ThrVal Thr Gln Gly Ser Gln 545 550 555 560 Met Asn Gly Ser Pro Lys Pro His565 19 490 PRT Drosophila melanogaster 19 Phe Arg Ser Ser Asn Asp TyrGly Val Asn Leu Gln Thr Ala Glu Met 1 5 10 15 Trp His His Thr Ile ArgLys His Lys Arg Gly Asn Gly Ser Ser Ser 20 25 30 Pro Ala Asp Cys Gly LysGln Leu Leu Ile Leu Leu Asn Pro Lys Ser 35 40 45 Gly Ser Gly Lys Gly ArgGlu Leu Phe Gln Lys Gln Val Ala Pro Leu 50 55 60 Leu Thr Glu Ala Glu ValGln Tyr Asp Leu Gln Ile Thr Thr His Pro 65 70 75 80 Gln Tyr Ala Lys GluPhe Val Arg Thr Arg Arg Asp Leu Leu Thr Arg 85 90 95 Tyr Ser Gly Ile ValVal Ala Ser Gly Asp Gly Leu Phe Tyr Glu Val 100 105 110 Leu Asn Gly LeuMet Glu Arg Met Asp Trp Arg Arg Ala Cys Arg Glu 115 120 125 Leu Pro LeuGly Ile Ile Pro Cys Gly Ser Gly Asn Gly Leu Ala Lys 130 135 140 Ser ValAla His His Cys Asn Glu Pro Tyr Glu Pro Lys Pro Ile Leu 145 150 155 160His Ala Thr Leu Thr Cys Met Ala Gly Lys Ser Thr Pro Met Asp Val 165 170175 Val Arg Val Glu Leu Ala Thr Arg Asp Lys His Phe Val Met Tyr Ser 180185 190 Phe Leu Ser Val Gly Trp Gly Leu Ile Ala Asp Ile Asp Ile Glu Ser195 200 205 Glu Arg Leu Arg Ser Ile Gly Ala Gln Arg Phe Thr Leu Trp AlaIle 210 215 220 Lys Arg Leu Ile Gly Leu Arg Ser Tyr Lys Gly Arg Val SerTyr Leu 225 230 235 240 Leu Gly Lys Gly Lys Lys Glu Pro Pro Val Glu AlaAla Arg Glu Leu 245 250 255 Pro Ala Glu Ser Thr Ala Ala Gly Ile Arg SerSer Leu Pro Leu Asn 260 265 270 Ala Gly Glu Phe His Asp Leu Pro Glu GluGlu Glu Gly Glu Ala Val 275 280 285 Leu Asp Gly Glu Gln Phe Ala Asp AlaIle Ser Leu Asp Arg Ser Val 290 295 300 Tyr Arg Gln His Ala Asp Ser TrpHis Ser Ala Met Ser Arg Arg Thr 305 310 315 320 Ala Tyr Tyr Ser Leu GlyGly Pro Ser Met Arg Ser Asn Arg Ser Arg 325 330 335 Met Ser Ile Ser GlnArg Ile Glu Ala Ala Asn Ala Glu Phe Ala Glu 340 345 350 Arg Val Pro ThrGly Thr Ile Pro Pro Leu Gln Met Pro Leu Leu Ser 355 360 365 Ser Asp GlyTrp Ile Cys Glu Asp Gly Asp Phe Val Met Val His Ala 370 375 380 Ala TyrThr Thr His Leu Ser Ser Asp Val Phe Phe Ala Pro Glu Ser 385 390 395 400Arg Leu Asp Asp Gly Leu Ile Tyr Leu Val Ile Ile Arg Arg Gly Val 405 410415 Ser Arg His Gln Leu Leu Asn Phe Met Leu Asn Leu Asn Ala Gly Thr 420425 430 His Leu Pro Ile Gly Glu Asp Pro Phe Ile Lys Val Val Pro Cys Arg435 440 445 Ala Phe Arg Ile Glu Pro Ser Ser Ser Asp Gly Ile Leu Val ValAsp 450 455 460 Gly Glu Arg Val Glu Tyr Gly Pro Ile Gln Ala Glu Val MetPro Gly 465 470 475 480 Leu Ile Asn Val Met Thr Thr Ser Gly Gln 485 49020 524 PRT Drosophila melanogaster 20 Phe Arg Ser Phe Asp Thr Phe GluAsp Asn Met Arg Glu Ala Asp Arg 1 5 10 15 Trp Tyr Arg Ser Leu Arg TrpGln Leu His Arg Thr Leu Glu Glu Ile 20 25 30 Phe Val Ala Pro Thr Val AspGlu Arg Arg Arg Arg Val Leu Val Leu 35 40 45 Leu Asn Pro Lys Ser Gly SerGly Asp Ala Arg Glu Val Phe Asn Met 50 55 60 His Val Thr Pro Val Leu AsnGlu Ala Glu Val Pro Tyr Asp Leu Tyr 65 70 75 80 Val Thr Lys His Ser AsnPhe Ala Ile Glu Phe Leu Ser Thr Arg Cys 85 90 95 Leu Asp Ala Trp Cys CysVal Val Ala Val Gly Gly Asp Gly Leu Phe 100 105 110 His Glu Ile Val AsnGly Leu Leu Gln Arg Gln Asp Trp Ala His Val 115 120 125 Leu Pro His LeuAla Leu Gly Ile Ile Pro Cys Gly Ser Gly Asn Gly 130 135 140 Leu Ala ArgSer Ile Ala His Cys Tyr Asn Lys Pro Val Leu Gly Ala 145 150 155 160 AlaLeu Thr Val Ile Ser Gly Arg Ser Ser Pro Met Asp Val Val Arg 165 170 175Val Gln Leu Gln Ser Arg Ser Leu Tyr Ser Phe Leu Ser Ile Gly Trp 180 185190 Gly Leu Ile Ser Asp Val Asp Ile Glu Ser Glu Arg Ile Arg Met Leu 195200 205 Gly Tyr Gln Arg Phe Thr Val Trp Thr Leu Tyr Arg Leu Val Asn Leu210 215 220 Arg Thr Tyr Asn Gly Arg Ile Ser Tyr Leu Leu Thr Asp His GluVal 225 230 235 240 Ser Ser Thr His Ser Ala Thr Gly Tyr Ala Ala Gln ArgArg Met Gln 245 250 255 Ser Ser Arg Ser Cys Asn Thr His Ile Asp Met LeuAsn Gly Pro Ala 260 265 270 Pro Ile Tyr His Ser Ser Ala Glu Tyr Leu ProGln Glu Phe Ala Asp 275 280 285 Val Ile Ser Leu Glu Thr Ser Ile Asn GlnSer Phe Arg Ser Arg Cys 290 295 300 Asp Ser Trp Leu Ser Gly Gly Ser ArgArg Ser Phe Tyr Tyr Ser Ile 305 310 315 320 Ser Glu Ser Ile Tyr His SerLeu Ala Asp Glu Ser Glu Phe Ala Gly 325 330 335 Leu Ala Ala Ala Ser LeuGlu Asn Arg Gln Gln Asn Tyr Gly Pro Ala 340 345 350 Ser Glu Leu Pro AspLeu Asn Glu Pro Leu Ser Glu Asp Gln Gly Trp 355 360 365 Leu Val Glu GluGly Glu Phe Val Met Met His Ala Val Tyr Gln Thr 370 375 380 His Leu GlyIle Asp Cys His Phe Ala Pro Lys Ala Gln Leu Asn Asp 385 390 395 400 GlyThr Ile Tyr Leu Ile Leu Ile Arg Ala Gly Ile Ser Arg Pro His 405 410 415Leu Leu Ser Phe Leu Tyr Asn Met Ser Ser Gly Thr His Leu Pro Glu 420 425430 Ser His Asp Asp His Val Lys Val Leu Pro Val Arg Ala Phe Arg Leu 435440 445 Glu Pro Tyr Asp Asn His Gly Ile Ile Thr Val Asp Gly Glu Arg Val450 455 460 Glu Phe Gly Pro Leu Gln Ala Glu Val Leu Pro Gly Ile Ala ArgVal 465 470 475 480 Met Val Pro Asn Val Ser Thr Phe Arg Phe Gln Ser AlaThr Leu Gln 485 490 495 His Gly Ile Pro Val Cys Ile Pro Val Arg Lys ArgPhe Val Leu Tyr 500 505 510 Asn Met Ser Ser Glu Glu Leu Ala Pro Ile AsnGlu 515 520 21 368 PRT Homo sapiens 21 Val Leu Val Leu Leu Asn Pro ArgGly Gly Lys Gly Lys Ala Leu Gln 1 5 10 15 Leu Phe Arg Ser His Val GlnPro Leu Leu Ala Glu Ala Glu Ile Ser 20 25 30 Phe Thr Leu Met Leu Thr GluArg Arg Asn His Ala Arg Glu Leu Val 35 40 45 Arg Ser Glu Glu Leu Gly ArgTrp Asp Ala Leu Val Val Met Ser Gly 50 55 60 Asp Gly Leu Met His Glu ValVal Asn Gly Leu Met Glu Arg Pro Asp 65 70 75 80 Trp Glu Thr Ala Ile GlnLys Pro Leu Cys Ser Leu Pro Ala Gly Ser 85 90 95 Gly Asn Ala Leu Ala AlaSer Leu Asn His Tyr Ala Gly Tyr Glu Gln 100 105 110 Val Thr Asn Glu AspLeu Leu Thr Asn Cys Thr Leu Leu Leu Cys Arg 115 120 125 Arg Leu Leu SerPro Met Asn Leu Leu Ser Leu His Thr Ala Ser Gly 130 135 140 Leu Arg LeuPhe Ser Val Leu Ser Leu Ala Trp Gly Phe Ile Ala Asp 145 150 155 160 ValAsp Leu Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu Met Arg Phe 165 170 175Thr Leu Gly Thr Phe Leu Arg Leu Ala Ala Leu Arg Thr Tyr Arg Gly 180 185190 Arg Leu Ala Tyr Leu Pro Val Gly Arg Val Gly Ser Lys Thr Pro Ala 195200 205 Ser Pro Val Val Val Gln Gln Gly Pro Val Asp Ala His Leu Val Pro210 215 220 Leu Glu Glu Pro Val Pro Ser His Trp Thr Val Val Pro Asp GluAsp 225 230 235 240 Phe Val Leu Val Leu Ala Leu Leu His Ser His Leu GlySer Glu Met 245 250 255 Phe Ala Ala Pro Met Gly Arg Cys Ala Ala Gly ValMet His Leu Phe 260 265 270 Tyr Val Arg Ala Gly Val Ser Arg Ala Met LeuLeu Arg Leu Phe Leu 275 280 285 Ala Met Glu Lys Gly Arg His Met Glu TyrGlu Cys Pro Tyr Leu Val 290 295 300 Tyr Val Pro Val Val Ala Phe Arg LeuGlu Pro Lys Asp Gly Lys Gly 305 310 315 320 Val Phe Ala Val Asp Gly GluLeu Met Val Ser Glu Ala Val Gln Gly 325 330 335 Gln Val His Pro Asn TyrPhe Trp Met Val Ser Gly Cys Val Glu Pro 340 345 350 Pro Pro Ser Trp LysPro Gln Gln Met Pro Pro Pro Glu Glu Pro Leu 355 360 365

What is claimed is:
 1. A recombinant expression vector comprising apolynucleotide as set forth in SEQ ID NO:15.
 2. A host cell transformedor transfected with an expression vector according to claim
 1. 3. Amethod for preparing a sphingosine-1-phosphate lyase, the methodcomprising culturing a host cell transformed or transfected with apolynucleotide according to claim 1 under conditions promotingexpression of the polynucleotide and recovering asphingosine-1-phosphate lyase.
 4. A method for identifying an agent thatmodulates sphingosine-1-phosphate lyase activity, comprising: (a)contacting a candidate agent with a polypeptide comprising an amino acidsequence selected from the group consisting of: (i) an amino acidsequence set forth in SEQ ID NO:16; (ii) an amino acid sequence havingat least 70% identity to a sequence set forth in SEQ ID NO:16; and (iii)an amino acid sequence having at least 90% identity to a sequence setforth in SEQ ID NO:16; wherein said polypeptide hassphingosine-1-phosphate lyase activity; and wherein the step ofcontacting is carried out under conditions and for a time sufficient toallow the candidate agent to interact with said polypeptide; and (b)subsequently measuring the ability of said polypeptide to degradesphingosine-1-phosphate or a derivative thereof, relative to an abilityin the absence of said candidate agent, and therefrom identifying anagent that modulates sphingosine-1-phosphate lyase activity.
 5. A methodaccording to claim 4, wherein the step of contacting is performed byincubating a cell expressing said polypeptide with the candidate agent,and wherein the step of measuring the ability to degradesphingosine-1-phosphate is performed using an in vitro assay and acellular extract.
 6. The method according to claim 5 wherein said cellhas been transformed or transfected with an expression vector accordingto claim
 1. 7. A pharmaceutical composition comprising an agent thatmodulates sphingosine-1-phosphate lyase activity of a polypeptidecomprising a sequence set forth in SEQ ID NO:16, in combination with apharmaceutically acceptable carrier.
 8. A composition according to claim7, wherein the agent comprises a polynucleotide.
 9. A compositionaccording to claim 7, wherein the agent comprises an antibody or anantigen-binding fragment thereof that specifically binds a sphingosinephosphate lyase (SPL) polypeptide comprising the sequence set forth inSEQ ID NO:16, and wherein the antibody increases the ability of the SPLpolypeptide to degrade sphingosine-1-phosphate.
 10. A method forinhibiting the growth of a cancer cell, comprising contacting saidcancer cell with an agent that increases sphingosine-1-phosphate lyaseactivity of a polypeptide comprising a sequence set forth in SEQ IDNO:16.
 11. A method according to claim 10, wherein the agent increasesexpression of an endogenous sphingosine-1-phosphate lyase gene.
 12. Amethod according to claim 11, wherein the agent comprises apolynucleotide set forth in SEQ ID NO:15.
 13. A method according toclaim 10, wherein the agent is capable of increasing the ability of apolypeptide comprising a sequence as set forth in SEQ ID NO:16 todegrade sphingosine-1-phosphate.
 14. A method according to claim 10,wherein the cancer cell is a breast cancer cell.
 15. A method forinhibiting the development, metastasis, or development and metastasis ofa cancer in a mammal, comprising administering to said mammal an agentthat increases sphingosine-1-phosphate lyase activity of a polypeptidecomprising a sequence set forth in SEQ ID NO:16.
 16. A method accordingto claim 15, wherein the agent increases expression of an endogenoussphingosine-1-phosphate lyase gene.
 17. A method according to claim 15,wherein the agent comprises a polynucleotide set forth in SEQ ID NO:15.18. A method according to claim 17, wherein the agent is capable ofincreasing the ability of a polypeptide comprising a sequence set forthin SEQ ID NO:16 to degrade sphingosine-1-phosphate.
 19. A methodaccording to claim 15, wherein the agent is linked to a targetingcomponent.
 20. A method according to claim 19, wherein the targetingcomponent is an anti-tumor antibody.
 21. A method according to claim 19,wherein the targeting component binds to an estrogen receptor.
 22. Amethod according to claim 15, wherein the mammal is afflicted withbreast cancer.
 23. An antibody or antigen-binding fragment thereof thatspecifically binds an sphingosine phosphate lyase (SPL) polypeptidecomprising the sequence set forth in SEQ ID NO:16, wherein the antibodyincreases the ability of the SPL polypeptide to degradesphingosine-1-phosphate.
 24. A method for detectingsphingosine-1-phosphate lyase in a sample, comprising: (a) contacting asample with an antibody according to claim 23 under conditions and for atime sufficient to allow the antibody to bind to sphingosine-1-phosphatelyase; and (b) detecting in the sample the presence ofsphingosine-1-phosphate lyase bound to the antibody.
 25. A kit fordetecting sphingosine-1-phosphate lyase in a sample, comprising anantibody according to claim 23 and a buffer and optionally a detectionreagent.
 26. A homozygous null mutant Drosophila melanogaster fly linethe genome of which comprises a P-element transposon insertion in thecoding region of the sphingosine phosphate lyase (SPL) gene wherein saidgene encodes the sequence set forth in SEQ ID NO:16, and wherein saidfly line has a flightless phenotype.
 27. A method for identifying anagent that modulates sphingosine-1-phosphate lyase activity, comprising:(a) culturing the mutant flies of claim 26 with growth mediasupplemented with a candidate agent under conditions and for a timesufficient to observe restoration of flight of at least a proportion ofsaid mutant flies; and (b) subsequently measuring the restoration offlight in said flies relative to the restoration of flight in theabsence of the candidate agent, and therefrom identifying an agent thatmodulates sphingosine-1-phosphate lyase activity.
 28. The methodaccording to claim 27 wherein said homozygous mutant fly line comprisesa sphingosine phosphate lyase (SPL) homozygous mutant fly line.
 29. Themethod according to claim 27 wherein said homozygous mutant fliesdemonstrate abnormal developmental patterning of thoracic muscles of theT2 segment.
 30. A method for determining the presence of a cancer in apatient, comprising the steps of: (a) obtaining a biological sample fromthe patient; (b) contacting the biological sample with at least oneoligonucleotide that is at least partially complementary to the sequenceset forth in SEQ ID NO:7; (c) detecting in the sample an amount of saidoligonucleotide that hybridizes to the polynucleotide; and (d) comparingthe amount of oligonucleotide that hybridizes to the polynucleotide to apredetermined cut-off value, and therefrom determining the presence ofthe cancer in the patient.